Bispecific EGFR/IGFIR binding molecules

ABSTRACT

The present invention relates to bispecific molecules comprising an EGFR binding domain and a distinct IGFIR binding domain for use in diagnostic, research and therapeutic applications. The invention further relates to cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and vectors comprising the polynucleotides encoding the innovative proteins. Exemplary bispecific molecules include antibody-like protein dimers based on the tenth fibronectin type III domain.

RELATED APPLICATIONS

This application claims the benefit of U.S. application Ser. No.12/625,217, filed Nov. 24, 2009, which claims benefit of U.S.Provisional Application Nos. 61/200,164, filed Nov. 24, 2008;61/200,282, filed Nov. 26, 2008; 61/212,966, filed Apr. 17, 2009;61/178,279, filed May 14, 2009; and 61/227,330, filed Jul. 21, 2009,which applications are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to EGFR binding domains and bispecificmolecules comprising an EGFR binding domain and a distinct IGFIR bindingdomain for use in diagnostic, research and therapeutic applications. Theinvention further relates to cells comprising such proteins,polynucleotide encoding such proteins or fragments thereof, and vectorscomprising the polynucleotides encoding the innovative proteins.Exemplary EGFR binding domains and bispecific molecules includeantibody-like protein dimers based on the tenth fibronectin type IIIdomain.

INTRODUCTION

Activation of receptor tyrosine kinase signaling is central to cancerdevelopment (see e.g., Grimberg A. Cancer Biol Ther. 2003 2(6):630-5 andMendelsohn J. J Clin Oncol. 2003 21(14):2787-99). Receptor tyrosinekinases have a conserved domain structure including an extracellulardomain, a transmembrane domain and an intracellular tyrosine kinasedomain. The extracellular domain can bind to a ligand, such as to apolypeptide growth factor or to a cell membrane-associated molecule.Typically, either ligand binding or ligand binding induced dimerizationof receptor tyrosine kinases activates the intracellular catalytictyrosine kinase domain of the receptor and subsequent signaltransduction.

Examples of receptor tyrosine kinases include, but are not limited toERBB receptors (e.g., EGFR, ERBB2, ERBB3, ERBB4),erythropoietin-producing hepatocellular (EPH) receptors, fibroblastgrowth factor (FGF) receptors (e.g., FGFR1, FGFR2, FGFR3, FGFR4, FGFR5),platelet-derived growth factor (PDGF) receptors (e.g., PDGFR-A,PDGFR-B), vascular endothelial growth factor (VEGF) receptors (e.g.,VEGFR1/FLT1, VEGFR2/FLK1, VEGF3), tyrosine kinase withimmunoglobulin-like and EGF-like domains (TIE) receptors, insulin-likegrowth factor (IGF) receptors (e.g., INS-R, IGFIR, IR-R), DiscoidinDomain (DD) receptors, receptor for c-Met (MET), recepteur d′ originenantais (RON); also known as macrophage stimulating 1 receptor, Flt3fins-related tyrosine kinase 3 (Flt3), colony stimulating factor 1(CSF1) receptor, adhesion related kinase receptor (e.g., Ax1), receptorfor c-kit (KIT) and insulin receptor related (IRR) receptors.

Inhibition of receptor tyrosine kinases has emerged as an effectivetreatment strategy for certain human malignancies (for a review seeRoussidis A E, In Vivo. 2002 16(6):459-69). While targeted monotherapymay initially be effective in treating cancer, therapeutic resistanceoften follows, possibly as a result of upregulation of other signalingcascades (see e.g., Nahta R et al., Breast Cancer Res. 2006 8(6):215 andHorn L et al., Clin Lung Cancer. 2007 8:S68-73). Accordingly, thereexists a need for developing improved cancer therapeutics.

SUMMARY OF THE INVENTION

In one aspect, the application provides EGFR binding tenth fibronectintype III domains (¹⁰Fn3) having novel sequences. EGFR binding ¹⁰Fn3having a consensus sequence are also provided. Such EGFR binding ¹⁰Fn3may be monomeric or may be included as part of a fusion protein.

In another aspect, the application provides bispecific molecules thatbind EGFR and IGFIR, referred to herein as “E/I binders”. E/I bindersencompassed by the invention include bispecific antibodies and dimers ofligand binding scaffold proteins (e.g., tendamistat, affibody,fibronectin type III domain, anticalin, tetranectin, and ankyrin). Whenconstructed as a single polypeptide chain, the E/I binders may beconstructed in any orientation, e.g., from N-terminus to C-terminuseither in the E-I arrangement or the I-E arrangement.

In one aspect, antibody-like protein dimers are provided comprising anEGFR binding ¹⁰Fn3 covalently or non-covalently linked to an IGFIRbinding ¹⁰Fn3. The ¹⁰Fn3 bind their target (EGFR or IGFIR) with a K_(D)of less than 500 nM. Each of the individual ¹⁰Fn3 independently has anamino acid sequence at least 70, 80, 85, 90, 95, 98, or 100% identicalto SEQ ID NO: 32, wherein n is an integer from 1-20, o is an integerfrom 1-20, and p is an integer from 1-40. In some embodiments, n is aninteger from 8-12, o is an integer from 4-8, and p is an integer from4-28. In some embodiments, n is 10, o is 6, and p is 12.

In some embodiments, the antibody-like protein dimers comprise IGFIRbinding ¹⁰Fn3 covalently linked to EGFR binding ¹⁰Fn3 via a polypeptidelinker or a polyethylene glycol moiety. In some embodiments, theantibody-like protein dimer comprises an amino acid sequence at least80, 90, 95, or 100% identical to any one of SEQ ID NOs: 20-31, 53-58,87-92, 98-105, 118-133, 149-154, 164-169, 179-184, 192-197, 205-210 and211-216.

In some embodiments, the E/I binder comprises an amino acid sequencehaving any one of SEQ ID NOs: 20-31, 53-58, 87-92, 98-105, 118-133,149-154, 164-169, 179-184, 192-197, 205-210 and 211-216, wherein (i) theEGFR binding ¹⁰Fn3 and/or the IGF-IR binding ¹⁰Fn3 comprises a ¹⁰Fn3scaffold having from has anywhere from 0 to 20, from 0 to 15, from 0 to10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 substitutions, conservative substitutions,deletions or additions relative to the corresponding scaffold aminoacids of SEQ ID NO: 1, and/or (ii) the EGFR binding ¹⁰Fn3 has anywherefrom 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions,conservative substitutions, deletions or additions relative to thecorresponding loop sequences of any one of SEQ ID NOs: 5-8, 52, 66-68,106-108, 112-114, 140-142, 155-157, 170-172, 182, 185-187, 198-200, or219-327 and/or the IGF-IR binding ¹⁰Fn3 has anywhere from 0 to 15, from0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to3, from 0 to 2, or from 0 to 1 substitutions, conservativesubstitutions, deletions or additions relative to the corresponding loopsequences of SEQ ID NO: 3.

In one aspect, pharmaceutically acceptable compositions are providedcomprising an antibody-like protein dimer as described herein and apharmaceutically acceptable carrier, wherein the composition isessentially pyrogen free.

In a further aspect, methods for treating hyperproliferative disorders,such as cancer, in a subject are provided comprising administering to asubject in need thereof a therapeutically effective amount of apharmaceutically acceptable composition comprising an antibody-likeprotein dimer as described herein.

In another aspect, the application provides a nucleic acid encoding anantibody-like protein dimer as described herein. Also provided is avector comprising a nucleic acid encoding an antibody-like dimer asdescribed herein. Suitable vectors include, for example, expressionvectors. Also provided are host cells comprising a nucleic, vector, orexpression vector, comprising a nucleic acid encoding an antibody-likeprotein dimer as described herein. Suitable host cells includeprokaryotic and eukaryotic host cells. Exemplary prokaryotic cells arebacterial cells, such as E. coli. Exemplary eukaryotic cells aremammalian cells, such as CHO cells. Also provided are methods forproducing an antibody-like protein dimer as described herein, comprisingculturing a host cell comprising a nucleic, vector, or expressionvector, comprising a nucleic acid encoding the antibody-like proteindimer and recovering the expressed antibody-like protein dimer from theculture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. SDS-PAGE Analysis of I1-GS10-E2. Samples from the lysis ofHMS174(DE3) bacterial cell pellet from which I1-GS10-E2 was expressedand purified by a HisTrap chromatography column were run on a 4-12%NuPAGE minigel and stained by Sypro-Orange and visualized by STORMimager. Mark 12 molecular weight standards (Lane 1); Lysate-soluble(Lane 2); Lysate-insoluble (Lane 3); HisTrap load (Lane 4); HisTrapnon-bound (Lane 5); Pooled HisTrap Eluate (Lane 6); Dialyzed into 50 mMNaOAc, 150 mM NaCl, pH 4.5 (Lane 7); Dialyzed into PBS (Lane 8);Dialyzed into Tris, 150 mM NaCl, pH 8.5 (Lane 9).

FIG. 2. A. SEC Analysis of midscale purified I1-GS10-E2. 22 μg ofHisTrap purified I1-GS10-E2 dialyzed into PBS, pH 7.4 was loaded onto aSuperdex 200 10/30 SEC Column (GE Healthcare) with a mobile phase of 100mM NaPO₄, 100 mM NaSO₄, 150 mM NaCl, pH 6.8 and measured using A280.I1-GS10-E2 eluted predominantly as a single monomeric species at amolecular weight range of approximately 24.6 kDa vs. globular GelFiltration standards (BioRad). B. SEC analysis of E2-GS10-I1.

FIG. 3. A. Differential Scanning calorimetry (DSC) of midscale purifiedI1-GS10-E2 in PBS was performed to determine the T_(m). A 1 mg/mLsolution of I1-GS10-E2 was scanned from 5° C. to 95° C. at a rate of 1degree per minute under 3 atm pressure. The data was analyzed versus acontrol run of the PBS buffer. B. DSC of E2-GS10-I1.

FIG. 4. Inhibition of IGFR activity in H292 cells. Cells were stimulatedwith 100 ng/mL of IGF-1 and 100 ng/mL of EGF and treated with either●I1, □E1, or ΔE1-GS10-I1 HTPP preparations. Phosphorylation of IGFIR ontyrosine 1131 was determined by ELISA.

FIG. 5. Inhibition of EGFR activity in H292 cells. Cells were stimulatedwith 100 ng/mL of IGF-1 and 100 ng/mL of EGF and treated with either●I1, □E1, or ΔE1-GS10-I1 HTPP preparations. Phosphorylation of EGFR ontyrosine 1068 was determined by ELISA.

FIG. 6. Inhibition of AKT phosphorylation in H292 cells. Cells werestimulated with 100 ng/mL of IGF-1 and 100 ng/mL of EGF and treated witheither ●I1, □E1, or ΔE1-GS10-I1 HTPP preparations. Phosphorylation ofAKT on serine 473 was determined by ELISA.

FIG. 7. Inhibition of RH41 cell proliferation. Cells were treated witheither ●I1, □E1, or ΔE1-GS10-I1 HTPP preparations and percent inhibitionof proliferation was determined.

FIG. 8. Inhibition of H292 cell proliferation. Cells were treated witheither ●I1, □E1, or ΔE1-GS10-I1 HTPP preparations and percent inhibitionof proliferation was determined.

FIG. 9. Summarizes IC50 values in cell based functional assays forisolated EGFR mononectins, E/I¹⁰ Fn3-based binders with serine at theC-terminal position without PEG added and E/I¹⁰ Fn3-based binders withcysteine at the C-terminal position conjugated to a 40 kDa branched PEG.Representative data is shown.

FIG. 10. Immunoblot analysis of PEGylated E/I¹⁰ Fn3-based binder with E2in the N-terminal and C-terminal positions. Despite both constructsdemonstrating comparable activity in the H292 cell assay for inhibitingEGFR, the E/I¹⁰ Fn3-based binder with E2 at the C-terminal position didnot degrade EGFR while the E/I¹⁰ Fn3-based binder with E2 at theN-terminal position did. Both constructs show very weak to no IGFRdegradation in this cell line. β-actin was included to demonstrate equalloading across all lanes. The phosphorylation state of EGFR, ERK and Shcwas also examined.

FIG. 11. Inhibition of EGF-stimulated EGFR phosphorylation in H292cells. Both constructs demonstrated comparable activity in the H292 cellassay for inhibiting EGFR. E2-GS10-I1 (with PEG) (◯), I1-GS10-E2 (withPEG) (□), panitumumab ( - - - ).

FIG. 12. Results of tumor xenograft studies. FIG. 12A: Preclinicalanti-tumor activity in the H292 human tumor xenograft model. Mean tumorsizes calculated from groups of 8 mice is shown in mg for controlanimals (▪), E3-GS10-I1 (w/PEG) dosed at 100 mg/kg (◯), E2-GS10-I1 (withPEG) dosed at 100 mg/kg (□), panitumumab dosed at 1 mg/mouse (Δ) or 0.1mg/mouse (∇). The letter a on the x-axis indicates doses of E/I bindersadministered and the p indicates doses of panitumumab administered. FIG.12B: Average weight change is shown for each group over the course ofthe study. Symbols are as described in FIG. 12A legend.

FIG. 13. Pharmacodynamic effects in the H292 NSCLC tumor xenograftmodel. Levels of the indicated analytes were determined in tumor lysatesas described in Example 12. (A) phosph-EGFR, (B) phospho-ErbB2, (C)phospho-IGFR, and (D) total EGFR. Checkered bars=panitumumab, emptybars=E2-GS10-I1 (with PEG), hatched bats=E3-GS10-I1 (with PEG).

FIG. 14. Western blot analysis of MCF7r cells compared to MCF7 parentalcells.

FIG. 15. MCF7 (Panel A) and MCF7r (Panel B) human tumor xenograftstudies in nude mice. Mean tumor size is shown for both studiescalculated from 8 mice per group.

FIG. 16. GEO human tumor xenograft studies in nude mice.

FIG. 17. H292 human tumor xenograft studies in nude mice.

FIG. 18. Colony formation assay with H292 NSCLC cells. A. Representativedata is shown from a single plate. B. IC50 from one E/I¹⁰ Fn3-basedbinder is shown with error bars calculated from triplicate measurements.

FIG. 19. Epitope mapping assay. Location of epitope binding for variousEGFR binding antibodies are shown in panel A. A description of theantibodies is provided in Example 18, Table 11. The left column of table11 provides a number for each anti-EGFR antibody which correlates withthe numbered antibodies shown in panel A. Panel B shows an exemplaryepitope mapping assay as described in Example 18.

FIG. 20. DSC analysis of the E/I¹⁰ Fn3-based binder, I1-GS10-E5pegylated, measured with a scan range of 15-95° C. at 1 mg/ml proteinconcentration in PBS, resulted in a Tm measurement of 55.2° C.

FIG. 21. Evaluation of E/I¹⁰ Fn3-based binders for inhibition of AKTphosphorylation in H292 cells as measured by ELISA. I1-GS10-E5-pegylated(◯) was more potent than I1-pegylated alone (▪) or E5-pegylated alone(▴) for blocking IGF1-stimulated AKT phosphorylation.

FIG. 22. Evaluation of E/I¹⁰ Fn3-based binders for inhibition of cellproliferation in H292 cells. I1-GS10-E5-pegylated (□) was more potentthan I1-pegylated alone (▴) and E5-pegylated alone (●) had only weakeffects for inhibiting the growth of H292 cells. Assays were carried outin triplicate. Representative data is shown.

FIG. 23. Evaluation of E/I¹⁰ Fn3-based binders for inhibition of cellproliferation in RH41 cells. I1-GS10-E5-pegylated (□) was slightly morepotent than I1-pegylated alone (▴) and E5-pegylated alone (●) orpanitumumab (dashed line) had almost no effect for inhibiting the growthof RH41 cells. Assays were carried out in triplicate. Representativedata is shown.

FIG. 24. Inhibition of ligand stimulated signaling by ¹⁰Fn3-basedbinders (pegylated). Effect of E/I¹⁰ Fn3-based binder (I1-GS10-E5pegylated) on receptor activation and cell signaling in DiFi (Panel A),H292 (Panel B) or BxPC3 (Panel C) cells. Cells were serum starved andtreated for 2 hours with 1 μM ¹⁰Fn3-based binders before stimulationwith either EGF, IGF1 or a combination of EGF+IGF1. GAPDH was probed toillustrate equal loading in all lanes.

FIG. 25. Inhibition of ligand stimulated signaling in H292 cells by¹⁰Fn3-based binders (unpegylated). Effect of E/I¹⁰ Fn3-based binder(E2-GS10-I1) on receptor activation and cell signaling in H292 cells.Cells were serum starved and treated for 2 hours with 1 μm ¹⁰Fn3-basedbinders before stimulation with either EGF, IGF1 or a combination ofEGF+IGF1. GAPDH was probed to illustrate equal loading in all lanes

FIG. 26. Competition binding studies with E/I¹⁰ Fn3-based binders. A.The EGFR ¹⁰Fn3-based binder does not compete for binding of EGFRantibodies to EGFR. Initial injection of the EGFR ¹⁰Fn3-based bindershows binding to EGFR on the surface of the chip. A second injection ofEGFR ¹⁰Fn3-based binder mixed with an equal amount of cetuximab,panitumumab, or nimotuzumab shows no competition for binding ofantibodies to EGFR by the EGFR ¹⁰Fn3-based binder. B. The E/I¹⁰Fn3-based binder can bind EGFR and IGF-IR simultaneously. Initialinjection of the E/I ¹⁰Fn3-based binder shows binding to EGFRimmobilized on the chip surface. A second injection of the E/I¹⁰Fn3-based binder soluble IGF-IR shows binding of sIGF-IR to other endof the immobilized E/I ¹⁰Fn3-based binder.

FIG. 27. TGFα plasma levels 4 hours after last dose of xenograftstudies. Plasma samples taken at the end of treatment from the BxPC3(Panel A), GEO (Panel B) and H441 (Panel C) xenograft studies describedin Table 24 were analyzed for circulating levels of TGFα.

FIG. 28. TGFα and IGF1 plasma levels in non tumor bearing nude miceafter dosing with I1-GS10-E5 pegylated. Non-tumor bearing mice weregiven a single dose of I1-GS10-E5 pegylated ¹⁰Fn3-based binder andanalyzed for circulating levels of TGFα (Panel A) and IGF1 (Panel B).

FIG. 29. H292 xenograft study using E/110Fn3-based binders as comparedto panitumumab. H292 xenografts were either untreated (▪) or dosed threetimes a week with ¹⁰Fn3-based binders formulated in PBS with theindividual constructs as described in the figure or dosed every threedays i.p. with panitumumab at 1 mg/mouse (◯) or 0.1 mg/mouse (□). Actualdoses of ¹⁰Fn3-based binders and panitumumab (▴) are indicated on thex-axis with the panitumumab doses closest to the x-axis below thetriangles indicating doses of ¹⁰Fn3-based binders. FIG. 29A showsmeasurements out to day 43. FIG. 29B shows measurements out to day 27.

FIG. 30. Pharmaokinectic parameters profile of E2-GS10-I1 pegylated inmice.

FIG. 31. Comparison of half-life at 100 mg/kg and 10 mg/kg IP, and 10mg/kg and 64 mg/kg SC in various E/I ¹⁰Fn3-based binders.

FIG. 32. Antitumor efficacy of E2-GS10-I1 pegylated in the RH41 model.

FIG. 33. Measurement of pharmacodynamic endpoints in tumors. At the endof treatment, tumors were removed 4 hours following the final dose fromDiFi xenograft model (panel A) and H292 xenograft model (panel B) andexamined for levels of phospho-EGFR, phospho-IGFR, total EGFR and totalIGFR. Equal amounts of total protein lysate was loaded into each lane ofthe gels and blots were also probed with GAPDH to demonstrate equalloading across all lanes.

FIG. 34. Sequence of anti-EGFR binder 679F09 (SEQ ID NO: 490). Loopresidues which were varied are underlined.

FIG. 35. BC loop Sequence Analysis I. Frequency of amino acids at eachposition in the BC loop from EGFR binding sequences. Image created usingWebLogo (Crooks G E, Hon G, Chandonia J M, Brenner S E. WebLogo: Asequence logo generator. Genome Research, 14:1188-1190, 2004).

FIG. 36. DE loop Sequence Analysis 1. Frequency of amino acids at eachposition in the DE loop from EGFR binding sequences (263 unique DE loopsequences analyzed).

FIG. 37. FG loop (10-aa length) Sequence Analysis I. Frequency of aminoacids at each position in the FG loop from EGFR binding sequences with10-amino acid long FG loops (228 unique 10-amino acid long FG loopsanalyzed).

FIG. 38. FG loop (15-aa length) Sequence Analysis I. Frequency of aminoacids at each position in the FG loop from EGFR binding sequences with15-amino acid long FG loops (349 unique 15-amino acid long FG loopsanalyzed).

FIG. 39. BC loop Sequence Analysis II. Frequency of amino acids at eachposition in the BC loop from all “potent” sequences (85 unique BC loopsequences analyzed).

FIG. 40. DE loop Sequence Analysis II. Frequency of amino acids at eachposition in the DE loop from all “potent” sequences (60 unique DE loopsequences analyzed).

FIG. 41. FG loop (10-aa length) Sequence Analysis II. Frequency of aminoacids at each position in the FG loop from all “potent” sequences with10-amino acid long FG loops (6 unique 10-amino acid long FG loopsanalyzed).

FIG. 42. FG loop (15-aa length) Sequence Analysis II. Frequency of aminoacids at each position in the FG loop from all “potent” sequences with15-amino acid long FG loops (65 unique 15-amino acid long FG loopsanalyzed).

FIG. 43. Table summarizing various characteristics of E/I ¹⁰Fn3-basedbinders as described in Example 22.

FIG. 44. Table summarizing various pharmacokinetic parameters of E/I¹⁰Fn3-based binders as described in Example 30.

FIGS. 45A-H. Amino acid sequences of E monomers as described in Example32. The BC, DE and FG loops in each sequence are underlined.

FIG. 46. Alignment of wild-type core sequence (amino acids 9-94 of SEQID NO: 1) with I1 core (SEQ ID NO:65), E1 core (SEQ ID NO:66), E2 core(SEQ ID NO:67), E3 core (SEQ ID NO:68), E4 core (SEQ ID NO:108), E5 core(SEQ ID NO:114), E85 core (SEQ ID NO:141), E90 core (SEQ ID NO:156), E96core (SEQ ID NO:171), E105 core (SEQ ID NO:186), and E112 core (SEQ IDNO:199). The BC, DE and FG loops in the wild-type sequences are shown inbold and underlined. The amino acid residues actually changed incomparison to wild-type for the I and E cores are shown i bold andunderlined.

FIGS. 47A-Q. Nucleic acid sequences of E and I monomers. Unlessotherwise specified, the nucleotide sequences encode a monomer having anN+10 N-terminal extension, a Ser tail, and a His tag.

FIGS. 48A-G. Nucleic acid sequence of E/I ¹⁰Fn3-based binders. Allnucleotide sequences encode an E/I ¹⁰Fn3-based binder having an N+10N-terminal extension on the first monomer in the construct and a Cystail and His tag on the second monomer in the construct. GS10 is SEQ IDNO: 11; GSGCGS8 is SEQ ID NO: 218; and GSGC is SEQ ID NO: 489.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “including” is used to mean “including but not limited to”.“Including” and “including but not limited to” are used interchangeably.

The term “antibody-like protein” refers to a non-immunoglobulin proteinhaving an “immunoglobulin-like fold”, i.e., comprises about 80-150 aminoacid residues that are structurally organized into a set of beta orbeta-like strands, forming beta sheets, where the beta or beta-likestrands are connected by intervening loop portions. The beta sheets formthe stable core of the antibody-like protein, while creating two “faces”composed of the loops that connect the beta or beta-like strands. Asdescribed herein, these loops can be varied to create customized ligandbinding sites, and such variations can be generated without disruptingthe overall stability of the protein. An example of such anantibody-like protein is a “fibronectin-based scaffold protein”, bywhich is meant a polypeptide based on a fibronectin type III domain(Fn3). In one aspect, an antibody-like protein is based on a tenthfibronectin type III domain (¹⁰Fn3).

By a “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thedisorder. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy in vivo can, for example, be measured by assessing the time todisease progression (TTP) and/or determining the response rates (RR).

The half-life of an amino acid sequence or compound can generally bedefined as the time taken for the serum concentration of the polypeptideto be reduced by 50% in vivo due to, e.g., degradation of the sequenceor compound and/or clearance or sequestration of the sequence orcompound by natural mechanisms. The half-life can be determined in anymanner known in the art, such as by pharmacokinetic analysis. See e.g.,M Gibaldi & D Perron “Pharmacokinetics”, published by Marcel Dekker, 2ndRev. edition (1982).

The term “E/I binder” refers to a bispecific molecule that comprises anEGFR binding domain and a distinct IGFIR binding domain. The two domainsmay be covalently or non-covalently linked. An exemplary E/I binder isan antibody-like dimer comprising an EGFR binding ¹⁰Fn3 and an IGFIRbinding ¹⁰Fn3, i.e., an E/I ¹⁰Fn3 based binder.

Overview

The epidermal growth factor receptor (EGFR) and insulin-like growthfactor receptor (IFGR) play key roles in the tumorigenesis of severaltypes of human cancer. Inhibition of either receptor effectively reducestumor growth in preclinical models as well as clinically. Blocking theEGFR pathway induces switching to the IGFR pathway to drive growth within vitro tumor models. Therefore, blocking both receptors simultaneouslymay achieve superior efficacy to blocking either pathway alone byovercoming pathway switching. In exemplary embodiments, the activity ofan E/I binder is synergistic in comparison to the monomeric componentsof the E/I binder.

The specification describes, inter alia, bispecific molecules that bindEGFR and IGFIR, referred to herein as “E/I binders”. Applicants havediscovered that such bispecific molecules inhibit proliferation of acancer model cell line with greater potency than the corresponding.monospecific binders (see e.g., Example 9 and FIG. 8).

E/I binders will be useful in numerous therapeutic applications,especially in the treatment of cancer. In addition to therapeuticapplications, E/I binders may be used in any circumstance where it isdesirable to detect EGFR and/or IGFIR.

E/I binders have an EGFR binding domain and a distinct IGFIR bindingdomain. Typical binding domains include antibodies; therefore,bispecific antibodies may be generated to function as E/I binders.Bispecific antibodies comprising complementary pairs of V_(H) and V_(L)regions are known in the art. These bispecific antibodies comprise twopairs of V_(H) and V_(L), each V_(H/L) pair binding to a single antigen.(see e.g., Hu et al., Cancer Res. 1996 56:3055-306; Neri et al., J. Mol.Biol. 1995 246:367-373; Atwell et al., Mol. Immunol. 1996 33:1301-1312;and Carter et al., Protein Sci. 1997 6:781-788). An exemplary bispecificantibody is a diabody, i.e., a small antibody fragment with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain connected to a light-chain variable domain in the samepolypeptide chain (Hollinger et al., Proc. Natl. Acad. Sci. USA 1993 90:6444-6448).

E/I binders also encompass dimers of ligand binding scaffold proteins.Scaffold proteins are well described in the literature and include,e.g., tendamistat, affibody, fibroncectin type III domain, anticalin,tetranectin, and ankyrin. Additional scaffold proteins that may be usedto generate E/I binders are reviewed in Binz et al., Nature Biotech23:1257-1268 (2005). Scaffold proteins are based on a rigid corestructure or ‘framework’ that is important in determining andstabilizing the three-dimensional structure. In between the fixed orconserved residues of the scaffold lie variable regions such as loops,surfaces or cavities that can be randomized to alter ligand binding. Alarge diversity of amino acids is provided in the variable regionsbetween the fixed scaffold residues to provide specific binding to atarget molecule.

An exemplary ligand binding scaffold protein is based on a fibronectintype III domain (Fn3). Fibronectin is a large protein which playsessential roles in the formation of extracellular matrix and cell-cellinteractions; it consists of many repeats of three types (types I, II,and III) of small domains.

Fn3 is small, monomeric, soluble, and stable. It lacks disulfide bondsand, therefore, is stable under reducing conditions. The overallstructure of Fn3 resembles the immunoglobulin fold. Fn3 domainscomprise, in order from N-terminus to C-terminus, a beta or beta-likestrand, A; a loop, AB; a beta or beta-like strand, B; a loop, BC; a betaor beta-like strand, C; a loop, CD; a beta or beta-like strand, D; aloop, DE; a beta or beta-like strand, E; a loop, EF; a beta or beta-likestrand, F; a loop, FG; and a beta or beta-like strand, G. The sevenantiparallel β-strands are arranged as two beta sheets that form astable core, while creating two “faces” composed of the loops thatconnect the beta or beta-like strands. Loops AB, CD, and EF are locatedat one face and loops BC, DE, and FG are located on the opposing face.Any or all of loops AB, BC, CD, DE, EF and FG may participate in ligandbinding. There are at least 15 different modules of Fn3, and while thesequence homology between the molecules is low, they all share a highsimilarity in tertiary structure.

Adnectins™ (Adnexus, a Bristol-Myers Squibb R&D Company) are ligandbinding scaffold proteins based on the tenth fibronectin type IIIdomain, i.e., the tenth module of Fn3, (¹⁰Fn3). The amino acid sequenceof a naturally occurring human ¹⁰Fn3 is set forth in SEQ ID NO: 1.

VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO:1) (BC, FG, and DEloops are emphasized)

In SEQ ID NO:1, the AB loop corresponds to residues 15-16, the BC loopcorresponds to residues 21-30, the CD loop corresponds to residues39-45, the DE loop corresponds to residues 51-56, the EF loopcorresponds to residues 60-66, and the FG loop corresponds to residues76-87. (Xu et al., Chemistry & Biology 2002 9:933-942). The BC, DE andFG loops align along one face of the molecule and the AB, CD and EFloops align along the opposite face of the molecule. In SEQ ID NO: 1,beta strand A corresponds to residues 9-14, beta strand B corresponds toresidues 17-20, beta strand C corresponds to residues 31-38, beta strandD corresponds to residues 46-50, beta strand E corresponds to residues57-59, beta strand F corresponds to residues 67-75, and beta strand Gcorresponds to residues 88-94. The strands are connected to each otherthrough the corresponding loop, e.g., strands A and B are connected vialoop AB in the formation strand A, loop AB, strand B, etc. Residuesinvolved in forming the hydrophobic core (the “core amino acidresidues”) include the amino acids corresponding to the following aminoacids of SEQ ID NO: 1: L8, V10, A13, L18, I20, W22, Y32, I34, Y36, F48,V50, A57, I59, L62, Y68, I70, V72, A74, I88, I90 and Y92, wherein thecore amino acid residues are represented by the single letter amino acidcode followed by the position at which they are located within SEQ IDNO: 1. See e.g., Dickinson et al., J. Mol. Biol. 236: 1079-1092 (1994).

As described above, amino acid residues corresponding to residues 21-30,51-56, and 76-87 of SEQ ID NO: 1 define the BC, DE and FG loops,respectively. However, it should be understood that not every residuewithin the loop region needs to be modified in order to achieve a ¹⁰Fn3binder having strong affinity for a desired target, such as IGF-IR orEGFR. For example, in many of the examples described herein, onlyresidues corresponding to amino acids 23-30, 52-55 and 77-86 of SEQ IDNO: 1 were modified to produce high affinity ¹⁰Fn3 binders (see FIG. 46.Accordingly, in certain embodiments, the BC loop may be defined by aminoacids corresponding to residues 23-30 of SEQ ID NO: 1, the DE loop maybe defined by amino acids corresponding to residues 52-55 of SEQ ID NO:1, and the FG loop may be defined by amino acids corresponding toresidues 77-86 of SEQ ID NO: 1.

¹⁰Fn3 are structurally and functionally analogous to antibodies,specifically the variable region of an antibody. While ¹⁰Fn3 domains maybe described as “antibody mimics” or “antibody-like proteins”, they dooffer a number of advantages over conventional antibodies. Inparticular, they exhibit better folding and thermostability propertiesas compared to antibodies, and they lack disulphide bonds, which areknown to impede or prevent proper folding under certain conditions.Exemplary E/I ¹⁰Fn3 based binders are predominantly monomeric with Tm'saveraging ˜50° C.

The BC, DE, and FG loops of ¹⁰Fn3 are analogous to the complementarydetermining regions (CDRs) from immunoglobulins. Alteration of the aminoacid sequence in these loop regions changes the binding specificity of¹⁰Fn3. The protein sequences outside of the CDR-like loops are analogousto the framework regions from immunoglobulins and play a role in thestructural conformation of the ¹⁰Fn3. Alterations in the framework-likeregions of ¹⁰Fn3 are permissible to the extent that the structuralconformation is not so altered as to disrupt ligand binding. Methods forgenerating ¹⁰Fn3 ligand specific binders have been described in PCTPublication Nos. WO 00/034787, WO 01/64942, and WO 02/032925, disclosinghigh affinity TNFα binders, PCT Publication No. WO 2008/097497,disclosing high affinity VEGFR2 binders, and PCT Publication No. WO2008/066752, disclosing high affinity IGFIR binders. Additionalreferences discussing ¹⁰Fn3 binders and methods of selecting bindersinclude PCT Publication Nos. WO 98/056915, WO 02/081497, and WO2008/031098 and U.S. Publication No. 2003186385.

Antibody-like proteins based on the ¹⁰Fn3 scaffold can be definedgenerally by the sequence:

VSDVPRDLEVVAATPTSLLI(X)_(n)YYRITYGETGGNSPVQEFTV(X)_(o)ATISGLKPGVDYTITVYAV(X)_(p)ISINYRT (SEQ ID NO: 32), wherein n is an integer from 1-20, ois an integer from 1-20, and p is an integer from 1-40. The BC, DE, andFG loops are represented by (X)_(n), (X)_(o), and (X)_(p), respectively.

¹⁰Fn3 generally begin with the amino acid residue corresponding tonumber 1 of SEQ ID NO: 1. However, domains with amino acid deletions arealso encompassed by the invention. In some embodiments, amino acidresidues corresponding to the first eight amino acids of SEQ ID NO: 1are deleted. Additional sequences may also be added to the N- orC-terminus. For example, an additional MG sequence may be placed at theN-terminus of ¹⁰Fn3. The M will usually be cleaved off, leaving a G atthe N-terminus. In some embodiments, sequences may be placed at theC-terminus of the ¹⁰Fn3 domain, e.g., EIDKPSQ (SEQ ID NO: 9), EIDKPCQ(SEQ ID NO: 10), EGSGS (SEQ ID NO: 96) or EGSGC (SEQ ID NO: 97).

The non-ligand binding sequences of ¹⁰Fn3, i.e., the “¹⁰Fn3 scaffold”,may be altered provided that the ¹⁰Fn3 retains ligand binding functionand/or structural stability. In some embodiments, one or more of Asp 7,Glu 9, and Asp 23 are replaced by another amino acid, such as, forexample, a non-negatively charged amino acid residue (e.g., Asn, Lys,etc.). These mutations have been reported to have the effect ofpromoting greater stability of the mutant ¹⁰Fn3 at neutral pH ascompared to the wild-type form (See, PCT Publication No. WO 02/04523). Avariety of additional alterations in the ¹⁰Fn3 scaffold that are eitherbeneficial or neutral have been disclosed. See, for example, Batori etal., Protein Eng. 2002 15(12):1015-20; Koide et al., Biochemistry 200140(34):10326-33.

The ¹⁰Fn3 scaffold may be modified by one or more conservativesubstitutions. As many as 5%, 10%, 20% or even 30% or more of the aminoacids in the ¹⁰Fn3 scaffold may be altered by a conservativesubstitution without substantially altering the affinity of the ¹⁰Fn3for a ligand. For example, the scaffold modification preferably reducesthe binding affinity of the ¹⁰Fn3 binder for a ligand by less than100-fold, 50-fold, 25-fold, 10-fold, 5-fold, or 2-fold. It may be thatsuch changes will alter the immunogenicity of the ¹⁰Fn3 in vivo, andwhere the immunogenicity is decreased, such changes will be desirable.As used herein, “conservative substitutions” are residues that arephysically or functionally similar to the corresponding referenceresidues. That is, a conservative substitution and its reference residuehave similar size, shape, electric charge, chemical properties includingthe ability to form covalent or hydrogen bonds, or the like. Preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al., Atlas of Protein Sequenceand Structure 5:345-352 (1978 & Supp.). Examples of conservativesubstitutions are substitutions within the following groups: (a) valine,glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d)aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine,threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,tyrosine.

E Binders

In one aspect, the disclosure provides antibody-like proteins comprisingan EGFR binding ¹⁰Fn3 domain. In certain embodiments, an EGFR binding¹⁰Fn3 may be provided as part of a fusion protein or multimer. Forexample, an EGFR binding ¹⁰Fn3 may be covalently or non-covalentlylinked to at least a second ¹⁰Fn3 binding domain. The second ¹⁰Fn3binding domain may bind to EGFR or to a different target. In anexemplary embodiment, an EGFR binding ¹⁰Fn3 may be covalently ornon-covalently linked to an IGF-IR binding ¹⁰Fn3.

In exemplary embodiments, the EGFR binding ¹⁰Fn3 proteins describedherein bind to EGFR with a K_(D) of less than 500 nM, 100 nM, 50 nM, 10nM, 1 nM, 500 pM, 100 pM, 100 pM, 50 pM or 10 pM.

In exemplary embodiments, the BC loop of the EGFR binding ¹⁰Fn3 proteinscorrespond to amino acids 23-30 of SEQ ID NO: 1, the DE loop of the EGFRbinding ¹⁰Fn3 proteins correspond to amino acids 52-55 of SEQ ID NO: 1,and the FG loop of the EGFR binding ¹⁰Fn3 proteins correspond to aminoacids 77-86 of SEQ ID NO: 1.

In one embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop having a YQ at the positions corresponding to aminoacids 29 and 30 of SEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop having a YQ at the positions corresponding to aminoacids 29 and 30 of SEQ ID NO: 1 and an FG loop that is fifteen aminoacids in length, e.g., an FG loop that is extended in length by fiveamino acids due to an insertion of five amino acids between residuescorresponding to amino acids 77-86 of SEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop having a YQ at the positions corresponding to aminoacids 29 and 30 of SEQ ID NO: 1 and a DE loop having a V, I, L, M or Aresidue at the position corresponding to amino acid 54 of SEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop having a YQ at the positions corresponding to aminoacids 29 and 30 of SEQ ID NO: 1, a DE loop having a V, I, L, M or Aresidue at the position corresponding to amino acid 54 of SEQ ID NO: 1,and an FG loop that is fifteen amino acids in length, e.g., an FG loopthat is extended in length by five amino acids due to an insertion offive amino acids between residues corresponding to amino acids 77-86 ofSEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop having a YQ at the positions corresponding to aminoacids 29 and 30 of SEQ ID NO: 1 and an FG loop comprising a D or N atthe position corresponding to amino acid 77 of SEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop having a YQ at the positions corresponding to aminoacids 29 and 30 of SEQ ID NO: 1 and an FG loop (i) that is fifteen aminoacids in length, e.g., an FG loop that is extended in length by fiveamino acids due to an insertion of five amino acids between residuescorresponding to amino acids 77-86 of SEQ ID NO: 1 and (ii) comprises aD or N at the position corresponding to amino acid 77 of SEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a DE loop comprising a V, I, L, M or A residue at theposition corresponding to amino acid 54 of SEQ ID NO: 1 and an FG loopcomprising a D or N at the position corresponding to amino acid 77 ofSEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a DE loop comprising a V, I, L, M or A residue at theposition corresponding to amino acid 54 of SEQ ID NO: 1 and an FG loop(i) that is fifteen amino acids in length, e.g., an FG loop that isextended in length by five amino acids due to an insertion of five aminoacids between residues corresponding to amino acids 77-86 of SEQ ID NO:1 and (ii) comprises a D or N at the position corresponding to aminoacid 77 of SEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop having a YQ at the positions corresponding to aminoacids 29 and 30 of SEQ ID NO: 1, a DE loop comprising a V, I, L, M or Aresidue at the position corresponding to amino acid 54 of SEQ ID NO: 1,and an FG loop comprising a D or N at the position corresponding toamino acid 77 of SEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop having a YQ at the positions corresponding to aminoacids 29 and 30 of SEQ ID NO: 1, a DE loop comprising a V, I, L, M or Aresidue at the position corresponding to amino acid 54 of SEQ ID NO: 1,and an FG loop (i) that is fifteen amino acids in length, e.g., an FGloop that is extended in length by five amino acids due to an insertionof five amino acids between residues corresponding to amino acids 77-86of SEQ ID NO: 1 and (ii) comprises a D or N at the positioncorresponding to amino acid 77 of SEQ ID NO: 1.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop comprising the amino acid sequence XXXXXXYQ, a DEloop comprising the amino acid sequence XX(V/I/L/M/A)X, and an FG loopcomprising the amino acid sequence (D/N)X_(n), wherein X is any aminoacid and n is 9-14 amino acids. In an exemplary embodiment, n is 14amino acids.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop corresponding to amino acids 23-30 of SEQ ID NO: 1comprising the amino acid sequence XXXXXXYQ, a DE loop corresponding toamino acids 52-55 of SEQ ID NO: 1 comprising the amino acid sequenceXX(V/I/L/M/A)X, and an FG loop corresponding to amino acids 77-86 of SEQID NO: 1 comprising the amino acid sequence (D/N)X_(n), wherein X is anyamino acid and n is 9-14 amino acids. In an exemplary embodiment, n is14 amino acids.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop comprising the amino acid sequence XXXXXXYQ, a DEloop comprising the amino acid sequence XX(V/I/L/M/A)X, and an FG loopcomprising an amino acid sequence selected from:

i. (D/N)(Y/M)(Y/A/M)(Y/H/F)(K/Q/V)(E/P/R)(Y/T/K)X(E/Y/Q)(Y/G/H); and

ii.D(Y/F/W)(Y/F/K)(N/D/P)(P/H/L)(A/T/V)(T/D/S)(H/Y/G)(E/P/V)(Y/H)(T/K/I)(Y/F)(H/N/Q)(T/Q/E)(T/S/I);

wherein X is any amino acid.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop comprising the amino acid sequence XXXXXXYQ, a DEloop comprising the amino acid sequence (G/Y/H)(D/M/G)(V/L/I)X, and anFG loop comprising an amino acid sequence(D/N)(Y/M)(Y/A/M)(Y/H/F)(K/Q/V)(E/P/R)(Y/T/K)X(E/Y/Q)(Y/G/H), wherein Xis any amino acid.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop comprising the amino acid sequence XXXXXXYQ, a DEloop comprising the amino acid sequence (G/Y/H)(D/M/G)(V/L/I)X, and anFG loop comprising an amino acid sequence D (Y/F/W) (Y/F/K)(N/D/P)(P/H/L)(A/T/V)(T/D/S)(H/Y/G)(E/P/V)(Y/H)(T/K/I)(Y/F)(H/N/Q)(T/Q/E)(T/S/I),wherein X is any amino acid.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop comprising the amino acid sequence XXXXXXYQ, a DEloop comprising the amino acid sequence XX(V/I/L/M/A)X, and an FG loopcomprising an amino acid sequence selected from:

(SEQ ID NO: 473) i. DY(A/Y)GKPYXEY; (SEQ ID NO: 474) ii.DY(A/Y)Y(K/R/Q/T)PYXEY; (SEQ ID NO: 475) iii.(D/N)Y(A/Y)(Y/F)(K/R/Q/T)EYXE(Y/H); (SEQ ID NO: 476) iv.DYY(H/Y)X(R/K)X(E/T)YX; (SEQ ID NO: 477) v.DYY(H/Y)(K/H/Q)(R/K)T(E/T)Y(G/P); (SEQ ID NO: 478) vi.(D/N)MMHV(E/D)YXEY; (SEQ ID NO: 479) vii. DYMHXXYXEY; and(SEQ ID NO: 480) viii. D(M/Y)YHX(K/R)X(V/I/L/M)YG;

wherein X is any amino acid.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop comprising the amino acid sequence XXXXXXYQ, a DEloop comprising the amino acid sequence XX(V/I/L/M/A)X, and an FG loopcomprising an amino acid sequence selected from:

(SEQ ID NO: 481) i. D(Y/F)(Y/F)NPXTHEYXYXXX; (SEQ ID NO: 482) ii.D(Y/F)(Y/F)D(P/L)X(T/S)HXYXYXXX; and (SEQ ID NO: 483) iii.D(Y/F)(K/R)PHXDGPH(T/I)YXE(S/Y);

wherein X is any amino acid.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop comprising the amino acid sequence XXXXXXYQ, a DEloop comprising the amino acid sequence XX(V/I/L/M/A)X, and an FG loopcomprising the amino acid sequence(DIN)(M/Y)(M/A/W)(H/F/Y)(V/K)EY(A/Q/R/S/T)E(Y/H/D), wherein X is anyamino acid.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop comprising the amino acid sequence XXXXXXYQ, a DEloop comprising the amino acid sequence XX(V/I/L/M/A)X, and an FG loopcomprising the amino acid sequenceD(Y/F/W)(Y/F/K)(N/P/D)(P/H/L)X(T/D/S)(H/G/Y)(E/P/Y)(Y/H)XYXXX, wherein Xis any amino acid.

In various embodiments, the DE loop of the EGFR binding ¹⁰Fn3 maycomprise the sequence (G/Y/H)(D/M/G)(V/L/I)X.

In another embodiment, the invention provides an EGFR binding ¹⁰Fn3comprising an FG loop comprising an amino acid sequence selected from:

(SEQ ID NO: 481) i. D(Y/F)(Y/F)NPXTHEYXYXXX; (SEQ ID NO: 482) ii.D(Y/F)(Y/F)D(P/L)X(T/S)HXYXYXXX; and (SEQ ID NO: 483) iii.D(Y/F)(K/R)PHXDGPH(T/I)YXE(S/Y);

wherein X is any amino acid.

In certain embodiments, the EGFR binding ¹⁰Fn3 comprises any of theconsensus sequences provided above, with the proviso that the EGFRbinding ¹⁰Fn3 does not comprise one or more of the following sequences:

(SEQ ID NO: 484) i. VSDVPRDLEVVAATPTSLLISWQVPRPMYQRYYRITYGETGGNSPVQEFTVPGGVRTATISGLKPGVDYTITVYAVTDYMHSEYRQ YPISINYRT, and(SEQ ID NO: 485) ii. VSDVPRDLEVVAATPTSLLISWQVPRPMYQYYRITYGETGGNSPVQEFTVPGGVRTATISGLKPGVDYTITVYAVTDYMHSEYRQY PISINYRT, and(SEQ ID NO: 486) iii. VSDVPRDLEVVAATPTSLLISWQVPRPMYQRYYRITYGETGGNSPVQEFTVPGGVRTATISGLKPGVDYTITVYAVTDYMHSEYRQ YPISINYRTEIDKPCQ.

In certain embodiments, an EGFR binding ¹⁰Fn3 comprising one of theconsensus sequences provided above has at least 40%, 50%, 60%, 70%, 75%,or 80% identity to SEQ ID NO: 1. In certain embodiments, the overallstructure of an EGFR binding ¹⁰Fn3 comprising one of the consensussequences provided above resembles the immunoglobulin fold. In certainembodiment, an EGFR binding ¹⁰Fn3 comprising one of the consensussequences provided above further comprises the core amino acid residuesof the scaffold. In certain embodiments, an EGFR binding ¹⁰Fn3comprising one of the consensus sequences provided above has at least70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one ofSEQ ID NOs: 5-8, 52, 66-68, 106-108, 112-114, 140-142, 155-157, 170-172,182, 185-187, 198-200, or 219-327. In certain embodiments, an EGFRbinding ¹⁰Fn3 comprising one of the consensus sequences provided abovehas at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity tothe amino acid sequence of amino acid residues corresponding to E9 ofSEQ ID NO: 1 through T94 of SEQ ID NO: 1 of any one of SEQ ID NOs: 5-8,52, 66-68, 106-108, 112-114, 140-142, 155-157, 170-172, 182, 185-187,198-200, or 219-327. In certain embodiments, the EGFR binding ¹⁰Fn3comprising one of the consensus sequences provided above comprises a¹⁰Fn3 scaffold having from has anywhere from 0 to 20, from 0 to 15, from0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to3, from 0 to 2, or from 0 to 1 substitutions, conservativesubstitutions, deletions or additions relative to the scaffold aminoacids residues of SEQ ID NO: 1.

In certain embodiments, the invention provides an EGFR binding ¹⁰Fn3comprising a BC loop having the amino acid sequence set forth in aminoacids 23-30, a DE loop having the amino acid sequence set forth in aminoacids 52-55, and an FG loop having the amino acid sequence set forth inamino acids 77-86 of any one of SEQ ID NOs: 219-327. In certainembodiments, the invention provides an EGFR binding ¹⁰Fn3 comprising aBC loop having the amino acid sequence set forth in amino acids 21-30, aDE loop having the amino acid sequence set forth in amino acids 51-56,and an FG loop having the amino acid sequence set forth in amino acids76-87 of any one of SEQ ID NOs: 219-327. In certain embodiments, theinvention provides an EGFR binding ¹⁰Fn3 comprising an amino acidsequence at least 60%, 75%, 80%, 85%, 90%, 95%, or 98% identical to anyone of SEQ ID NOs: 219-327.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 21-30 of SEQ ID NO: 5, a DE loophaving the amino acid sequence set forth in amino acids 51-56 of SEQ IDNO: 5, and an FG loop having the amino acid sequence set forth in aminoacids 76-92 of SEQ ID NO: 5. In some embodiments, the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence X_(g)DSGRGSYQX_(h)(SEQ ID NO: 40), a DE loop having the amino acid sequence X_(i)GPVHX_(j)(SEQ ID NO: 42), and an FG loop having the amino acid sequenceX_(k)DHKPHADGPHTYHEX_(l) (SEQ ID NO: 44); wherein X is any amino acidand g, h, i, j, k, and l are integers independently selected from 0 to5. In some embodiments, the EGFR binding ¹⁰Fn3 comprises a BC loophaving the amino acid sequence SWDSGRGSYQ (SEQ ID NO: 39), a DE loophaving the amino acid sequence PGPVHT (SEQ ID NO: 41), and an FG loophaving the amino acid sequence TDHKPHADGPHTYHESP (SEQ ID NO: 43). Insome embodiments, the EGFR binding ¹⁰Fn3 has an amino acid sequence atleast 80, 90, 95, or 100% identical to SEQ ID NOs: 5 or 6.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 21-30 of SEQ ID NO: 7, a DE loophaving the amino acid sequence set forth in amino acids 51-56 of SEQ IDNO: 7, and an FG loop having the amino acid sequence set forth in aminoacids 76-87 of SEQ ID NO: 7. In some embodiments, the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence X_(m)VAGAEDYQX_(n)(SEQ ID NO: 34), a DE loop having the amino acid sequence X_(o)HDLVX_(p)(SEQ ID NO: 36), and an FG loop having the amino acid sequenceX_(q)DMMHVEYTEHX_(r) (SEQ ID NO: 38); wherein X is any amino acid and m,n, o, p, q, and r are integers independently selected from 0 to 5. Insome embodiments, the EGFR binding ¹⁰Fn3 comprises a BC loop having theamino acid sequence SWVAGAEDYQ (SEQ ID NO: 33), a DE loop having theamino acid sequence PHDLVT (SEQ ID NO: 35), and an FG loop having theamino acid sequence TDMMHVEYTEHP (SEQ ID NO: 37). In some embodiments,the EGFR binding ¹⁰Fn3 has an amino acid sequence at least 80, 90, 95,or 100% identical to SEQ ID NO: 7 or 8.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 23-30 of SEQ ID NO: 82, a DE loophaving the amino acid sequence set forth in amino acids 51-55 of SEQ IDNO: 82, and an FG loop having the amino acid sequence set forth in aminoacids 76-86 of SEQ ID NO: 82. In some embodiments, the EGFR binding¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(s)LPGKLRYQX_(t) (SEQ ID NO: 60), a DE loop having the amino acidsequence X_(u)HDLR_(w) (SEQ ID NO: 62), and an FG loop having the aminoacid sequence X_(y)NMMHVEYSEYX_(z) (SEQ ID NO: 64); wherein X is anyamino acid and s, t, u, w, y and z are integers independently selectedfrom 0 to 5. In some embodiments, the EGFR binding ¹⁰Fn3 comprises a BCloop having the amino acid sequence LPGKLRYQ (residues 3-13 of SEQ IDNO: 59), a DE loop having the amino acid sequence PHDLR (residues 1-5 ofSEQ ID NO: 61), and an FG loop having the amino acid sequenceTNMMHVEYSEY (residues 1-11 of SEQ ID NO: 63). In some embodiments, theEGFR binding ¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, or100% identical to SEQ ID NO: 52 or 82.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 23-30 of SEQ ID NO: 106, a DE loophaving the amino acid sequence set forth in amino acids 51-55 of SEQ IDNO: 106, and an FG loop having the amino acid sequence set forth inamino acids 76-86 of SEQ ID NO: 106. In some embodiments, the EGFRbinding ¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(g)HERDGSRQX_(h) (SEQ ID NO: 134), a DE loop having the amino acidsequence X_(i)GGVRX_(j) (SEQ ID NO: 135), and an FG loop having theamino acid sequence X_(k)DYFNPTTHEYIYQTTX_(l) (SEQ ID NO: 136); whereinX is any amino acid and g, h, i, j, k and l are integers independentlyselected from 0 to 5. In some embodiments, the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence SWHERDGSRQ (SEQ IDNO: 109), a DE loop having the amino acid sequence PGGVRT (SEQ ID NO:110), and an FG loop having the amino acid sequence TDYFNPTTHEYIYQTTP(SEQ ID NO: 111). In some embodiments, the EGFR binding ¹⁰Fn3 has anamino acid sequence at least 80, 90, 95, or 100% identical to any one ofSEQ ID NOs: 106-108.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 23-30 of SEQ ID NO: 112, a DE loophaving the amino acid sequence set forth in amino acids 51-55 of SEQ IDNO: 112, and an FG loop having the amino acid sequence set forth inamino acids 76-86 of SEQ ID NO: 112. In some embodiments, the EGFRbinding ¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(g)WAPVDRYQX_(h) (SEQ ID NO: 137), a DE loop having the amino acidsequence X_(i)RDVYX_(j) (SEQ ID NO: 138), and an FG loop having theamino acid sequence X_(k)DYKPHADGPHTYHESX_(l) (SEQ ID NO: 139); whereinX is any amino acid and g, h, i, j, k and l are integers independentlyselected from 0 to 5. In some embodiments, the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence SWWAPVDRYQ (SEQ IDNO: 115), a DE loop having the amino acid sequence PRDVYT (SEQ ID NO:116), and an FG loop having the amino acid sequence TDYKPHADGPHTYHESP(SEQ ID NO: 117). In some embodiments, the EGFR binding ¹⁰Fn3 has anamino acid sequence at least 80, 90, 95, or 100% identical to any one ofSEQ ID NOs: 112-114.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 13-22 of SEQ ID NO: 141, a DE loophaving the amino acid sequence set forth in amino acids 43-48 of SEQ IDNO: 141, and an FG loop having the amino acid sequence set forth inamino acids 68-84 of SEQ ID NO: 141. In some embodiments, the EGFRbinding ¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(g)TQGSTHYQX_(h) (SEQ ID NO: 146), a DE loop having the amino acidsequence X_(i)GMVYX_(j) (SEQ ID NO: 147), and an FG loop having theamino acid sequence X_(k)DYFDRSTHEYKYRTTX_(l) (SEQ ID NO: 148); whereinX is any amino acid and g, h, i, j, k and l are integers independentlyselected from 0 to 5. In some embodiments, the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence SWTQGSTHYQ (SEQ IDNO: 143), a DE loop having the amino acid sequence PGMVYT (SEQ ID NO:144), and an FG loop having the amino acid sequence TDYFDRSTHEYKYRTTP(SEQ ID NO: 145). In some embodiments, the EGFR binding ¹⁰Fn3 has anamino acid sequence at least 80, 90, 95, or 100% identical to any one ofSEQ ID NOs: 140-142.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 13-22 of SEQ ID NO: 156, a DE loophaving the amino acid sequence set forth in amino acids 43-48 of SEQ IDNO: 156, and an FG loop having the amino acid sequence set forth inamino acids 68-84 of SEQ ID NO: 156. In some embodiments, the EGFRbinding ¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(g)YWEGLPYQX_(h) (SEQ ID NO: 161), a DE loop having the amino acidsequence X_(i)RDVNX_(j) (SEQ ID NO: 162), and an FG loop having theamino acid sequence X_(k)DWYNPDTHEYIYHTIX_(l) (SEQ ID NO: 163); whereinX is any amino acid and g, h, i, j, k and l are integers independentlyselected from 0 to 5. In some embodiments, the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence SWYWEGLPYQ (SEQ IDNO: 158), a DE loop having the amino acid sequence PRDVNT (SEQ ID NO:159), and an FG loop having the amino acid sequence TDWYNPDTHEYIYHTIP(SEQ ID NO: 160). In some embodiments, the EGFR binding ¹⁰Fn3 has anamino acid sequence at least 80, 90, 95, or 100% identical to any one ofSEQ ID NOs: 155-157.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 13-22 of SEQ ID NO: 171, a DE loophaving the amino acid sequence set forth in amino acids 43-48 of SEQ IDNO: 171, and an FG loop having the amino acid sequence set forth inamino acids 68-84 of SEQ ID NO: 171. In some embodiments, the EGFRbinding ¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(g)ASNRGTYQX_(h) (SEQ ID NO: 176), a DE loop having the amino acidsequence X_(i)GGVSX_(j) (SEQ ID NO: 177), and an FG loop having theamino acid sequence X_(k)DAFNPTTHEYNYFTTX_(l) (SEQ ID NO: 178); whereinX is any amino acid and g, h, i, j, k and l are integers independentlyselected from 0 to 5. In some embodiments, the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence SWASNRGTYQ (SEQ IDNO: 173), a DE loop having the amino acid sequence PGGVST (SEQ ID NO:174), and an FG loop having the amino acid sequence TDAFNPTTHEYNYFTTP(SEQ ID NO: 175). In some embodiments, the EGFR binding ¹⁰Fn3 has anamino acid sequence at least 80, 90, 95, or 100% identical to any one ofSEQ ID NOs: 170-172.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 13-22 of SEQ ID NO: 186, a DE loophaving the amino acid sequence set forth in amino acids 43-48 of SEQ IDNO: 186, and an FG loop having the amino acid sequence set forth inamino acids 68-84 of SEQ ID NO: 186. In some embodiments, the EGFRbinding ¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(g)DAPTSRYQX_(h) (SEQ ID NO: 190), a DE loop having the amino acidsequence X_(i)GGLSX_(j) (SEQ ID NO: 191), and an FG loop having theamino acid sequence X_(k)DYKPHADGPHTYHESX₁ (SEQ ID NO: 139); wherein Xis any amino acid and g, h, i, j, k and l are integers independentlyselected from 0 to 5. In some embodiments, the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence SWDAPTSRYQ (SEQ IDNO: 188), a DE loop having the amino acid sequence PGGLST (SEQ ID NO:189), and an FG loop having the amino acid sequence TDYKPHADGPHTYHESP(SEQ ID NO: 117). In some embodiments, the EGFR binding ¹⁰Fn3 has anamino acid sequence at least 80, 90, 95, or 100% identical to any one ofSEQ ID NOs: 185-187.

In one embodiment, an antibody-like protein is provided comprising atenth fibronectin type III domain (¹⁰Fn3) that binds EGFR with a K_(D)of less than 500 nM and comprises a BC loop having the amino acidsequence set forth in amino acids 13-22 of SEQ ID NO: 199, a DE loophaving the amino acid sequence set forth in amino acids 43-48 of SEQ IDNO: 199, and an FG loop having the amino acid sequence set forth inamino acids 68-84 of SEQ ID NO: 199. In some embodiments, the EGFRbinding ¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(g)DAGAVTYQX_(h) (SEQ ID NO: 203), a DE loop having the amino acidsequence X_(i)GGVRX_(j) (SEQ ID NO: 135), and an FG loop having theamino acid sequence X_(k)DYKPHADGPHTYHEYX_(l) (SEQ ID NO: 204); whereinX is any amino acid and g, h, i, j, k and l are integers independentlyselected from 0 to 5. In some embodiments, the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence SWDAGAVTYQ (SEQ IDNO: 201), a DE loop having the amino acid sequence PGGVRT (SEQ ID NO:110), and an FG loop having the amino acid sequence TDYKPHADGPHTYHEYP(SEQ ID NO: 202). In some embodiments, the EGFR binding ¹⁰Fn3 has anamino acid sequence at least 80, 90, 95, or 100% identical to any one ofSEQ ID NOs: 198-200.

In certain embodiments, an EGFR binding ¹⁰Fn3 domain is covalently ornon-covalently linked to an EGF-IR binding ¹⁰Fn3 domain. In exemplaryembodiments, the IGF-IR binding ¹⁰Fn3 may comprise a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 76-83 of SEQ ID NO: 3. In some embodiments, the IGF-IRbinding ¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having the amino acidsequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loop having the aminoacid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50), wherein X is any aminoacid and a, b, c, d, e, f, g, h, i, j, k, and l are integersindependently selected from 0 to 5, or wherein a is 2 and b-f are 1, orwherein a-f are zero. In some embodiments, the IGF-IR binding ¹⁰Fn3 hasan amino acid sequence at least 80, 90, 95, 98, 99, or 100% identical toSEQ ID NO: 3. In certain embodiments, the IGF-IR binding ¹⁰Fn3 comprisesa ¹⁰Fn3 scaffold having from has anywhere from 0 to 20, from 0 to 15,from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0to 3, from 0 to 2, or from 0 to 1 substitutions, conservativesubstitutions, deletions or additions relative to the scaffold aminoacid residues of SEQ ID NO: 1. In certain embodiments, the IGF-IRbinding ¹⁰Fn3 has anywhere from 0 to 15, from 0 to 10, from 0 to 8, from0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to1 substitutions, conservative substitutions, deletions or additionsrelative to the corresponding loop sequences of SEQ ID NO: 3.

¹⁰Fn3 E/I Binders

One aspect of the disclosure provides E/I binders constructed fromantibody-like protein multimers. In some embodiments, an antibody-likeprotein multimer comprises at least one EGFR binding ¹⁰Fn3 covalently ornon-covalently linked to at least one IGFIR binding ¹⁰Fn3. In certainembodiments, the E/I binders described herein may be constructed as asingle polypeptide chain wherein the E and I subunits may be in eitherorientation, e.g., from N-terminus to C-terminus, in the E-I orientationor in the I-E orientation.

The disclosure relates, in part, to the surprising discovery thatmultiple ¹⁰Fn3 joined via a polypeptide linker correctly foldindependently of each other, retain high affinity binding, and that eachof the domains retains its functional properties (see e.g., Examples5-10). Additionally, these E/I ¹⁰Fn3 based binders demonstrate desirablebiophysical properties such as low aggregation and high meltingtemperature (T_(m)) (see e.g., Example 4). The Examples characterize avariety of E/I ¹⁰Fn3 based binders. An exemplary IGFIR binding ¹⁰Fn3 isset forth in SEQ ID NO: 4. Exemplary EGFR binding ¹⁰Fn3 are set forth inSEQ ID NOs: 6, 8, 52, 107, 113, 140, 155, 170, 185 and 198.

In some embodiments, an E/I binder comprises an EGFR binding ¹⁰Fn3 andan IGFIR binding ¹⁰Fn3, independently having an amino acid sequence atleast 40, 50, 60, 70, or 80% identical to the human ¹⁰Fn3 domain, shownin SEQ ID NO: 1. Much of the variability will generally occur in one ormore of the loops.

In some embodiments, an E/I binder comprises an EGFR binding ¹⁰Fn3 andan IGFIR binding ¹⁰Fn3, independently having an amino acid sequence atleast 70, 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 32,wherein n is an integer from 1-20, o is an integer from 1-20, and p isan integer from 1-40. In some embodiments, n is an integer from 8-12, ois an integer from 4-8, and p is an integer from 4-28. In someembodiments, n is 10, o is 6, and p is 12.

In some embodiments, the disclosure provides multimers of ¹⁰Fn3 havingat least one loop selected from loop BC, DE, and FG with an alteredamino acid sequence relative to the sequence of the corresponding loopof the human ¹⁰Fn3. By “altered” is meant one or more amino acidsequence alterations relative to a template sequence (correspondinghuman fibronectin domain) and includes amino acid additions, deletions,and substitutions. Altering an amino acid sequence may be accomplishedthrough intentional, blind, or spontaneous sequence variation, generallyof a nucleic acid coding sequence, and may occur by any technique, forexample, PCR, error-prone PCR, or chemical DNA synthesis. In someembodiments, an amino acid sequence is altered by substituting with oradding naturally occurring amino acids.

In some embodiments, one or more loops selected from BC, DE, and FG maybe extended or shortened in length relative to the corresponding humanfibronectin loop. In particular, the FG loop of the human ¹⁰Fn3 is 12residues long, whereas the corresponding loop in antibody heavy chainsranges from 4-28 residues. To optimize antigen binding, therefore, thelength of the FG loop of ¹⁰Fn3 may be altered in length as well as insequence to obtain the greatest possible flexibility and affinity inantigen binding.

In some embodiments of the ¹⁰Fn3 molecules, the altered BC loop has upto 10 amino acid substitutions, up to 9 amino acid deletions, up to 10amino acid insertions, or a combination of substitutions and deletionsor insertions. In some embodiments, the altered DE loop has up to 6amino acid substitutions, up to 5 amino acid deletions, up to 14 aminoacid insertions or a combination of substitutions and deletions orinsertions. In some embodiments, the FG loop has up to 12 amino acidsubstitutions, up to 11 amino acid deletions, up to 28 amino acidinsertions or a combination of substitutions and deletions orinsertions.

Naturally occurring ¹⁰Fn3 comprises an “arginine-glycine-aspartic acid”(RGD) integrin-binding motif in the FG loop. Preferred multimers of¹⁰Fn3 lack an RGD integrin-binding motif.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 5, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 5, and an FG loop having the amino acid sequence set forth inamino acids 76-92 of SEQ ID NO: 5; covalently or non-covalently linkedto b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having the amino acidsequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DE loophaving the amino acid sequence set forth in amino acids 51-56 of SEQ IDNO: 3, and an FG loop having the amino acid sequence set forth in aminoacids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding ¹⁰Fn3has an amino acid sequence at least 80, 90, 95, 98, 99, or 100%identical to SEQ ID NO: 5. In some embodiments, the IGFIR binding ¹⁰Fn3has an amino acid sequence at least 80, 90, 95, 98, 99, or 100%identical to SEQ ID NO: 3. In some embodiments, the E/I binder comprisesan amino acid sequence at least 80, 85, 90, 95, 98, 99, or 100%identical to SEQ ID NOs: 20, 21, 23, 24, 90, 92, 101 or 103.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 7, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 7, and an FG loop having the amino acid sequence set forth inamino acids 76-87 of SEQ ID NO: 7; covalently or non-covalently linkedto b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having the amino acidsequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DE loophaving the amino acid sequence set forth in amino acids 51-56 of SEQ IDNO: 3, and an FG loop having the amino acid sequence set forth in aminoacids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding ¹⁰Fn3has an amino acid sequence at least 80, 90, 95, 98, or 100% identical toSEQ ID NO: 7. In some embodiments, the IGFIR binding ¹⁰Fn3 has an aminoacid sequence at least 80, 90, 95, 98, or 100% identical to SEQ ID NO:3. In some embodiments, the E/I binder comprises an amino acid sequenceat least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NOs: 26, 27,29, 30, 89, 91, 100 or 102.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 82, aDE loop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 82, and an FG loop having the amino acid sequence set forthin amino acids 76-87 of SEQ ID NO: 82; covalently or non-covalentlylinked to b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 82. In some embodiments, the IGFIR binding ¹⁰Fn3has an amino acid sequence at least 80, 90, 95, 98, or 100% identical toSEQ ID NO: 3. In some embodiments, the E/I binder comprises an aminoacid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ IDNOs: 53, 54, 87, 88, 98, 99, 104 or 105.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 106, aDE loop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 106, and an FG loop having the amino acid sequence set forthin amino acids 76-92 of SEQ ID NO: 106; covalently or non-covalentlylinked to b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 106. In some embodiments, the IGFIR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 3. In some embodiments, the E/I binder comprisesan amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical toSEQ ID NOs: 118-125.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 112, aDE loop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 112, and an FG loop having the amino acid sequence set forthin amino acids 76-92 of SEQ ID NO: 112; covalently or non-covalentlylinked to b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 112. In some embodiments, the IGFIR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 3. In some embodiments, the E/I binder comprisesan amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical toSEQ ID NOs: 126-133.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 13-22 of SEQ ID NO: 141, aDE loop having the amino acid sequence set forth in amino acids 43-48 ofSEQ ID NO: 141, and an FG loop having the amino acid sequence set forthin amino acids 68-84 of SEQ ID NO: 141; covalently or non-covalentlylinked to b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 140, 141, 142 or 300. In some embodiments, theIGFIR binding ¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98,or 100% identical to SEQ ID NO: 3. In some embodiments, the E/I bindercomprises an amino acid sequence at least 80, 85, 90, 95, 98, or 100%identical to SEQ ID NOs: 149-154.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 13-22 of SEQ ID NO: 156, aDE loop having the amino acid sequence set forth in amino acids 43-48 ofSEQ ID NO: 156, and an FG loop having the amino acid sequence set forthin amino acids 68-84 of SEQ ID NO: 156; covalently or non-covalentlylinked to b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 155, 156, 157 or 305. In some embodiments, theIGFIR binding ¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98,or 100% identical to SEQ ID NO: 3. In some embodiments, the E/I bindercomprises an amino acid sequence at least 80, 85, 90, 95, 98, or 100%identical to SEQ ID NOs: 158-166.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 13-22 of SEQ ID NO: 171, aDE loop having the amino acid sequence set forth in amino acids 43-48 ofSEQ ID NO: 171, and an FG loop having the amino acid sequence set forthin amino acids 68-84 of SEQ ID NO: 171; covalently or non-covalentlylinked to b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 170, 171, 172 or 311. In some embodiments, theIGFIR binding ¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98,or 100% identical to SEQ ID NO: 3. In some embodiments, the E/I bindercomprises an amino acid sequence at least 80, 85, 90, 95, 98, or 100%identical to SEQ ID NOs: 179-184.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 13-22 of SEQ ID NO: 186, aDE loop having the amino acid sequence set forth in amino acids 43-48 ofSEQ ID NO: 186, and an FG loop having the amino acid sequence set forthin amino acids 68-84 of SEQ ID NO: 186; covalently or non-covalentlylinked to b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 185, 186, 187 or 320. In some embodiments, theIGFIR binding ¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98,or 100% identical to SEQ ID NO: 3. In some embodiments, the E/I bindercomprises an amino acid sequence at least 80, 85, 90, 95, 98, or 100%identical to SEQ ID NOs: 192-197.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an EGFR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 13-22 of SEQ ID NO: 199, aDE loop having the amino acid sequence set forth in amino acids 43-48 ofSEQ ID NO: 199, and an FG loop having the amino acid sequence set forthin amino acids 68-84 of SEQ ID NO: 199; covalently or non-covalentlylinked to b) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 21-30 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 51-56 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98, or 100%identical to SEQ ID NO: 198, 199, 200 or 327. In some embodiments, theIGFIR binding ¹⁰Fn3 has an amino acid sequence at least 80, 90, 95, 98,or 100% identical to SEQ ID NO: 3. In some embodiments, the E/I bindercomprises an amino acid sequence at least 80, 85, 90, 95, 98, or 100%identical to SEQ ID NOs: 205-210.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(g)DSGRGSYQX_(h) (SEQ ID NO:40), a DE loop having the amino acid sequence X_(i)GPVHX_(j) (SEQ ID NO:42), and an FG loop having the amino acid sequenceX_(k)DHKPHADGPHTYHEX_(l)(SEQ ID NO: 44); wherein X is any amino acid anda, b, c, d, e, f, g, h, i, j, k, and l are integers independentlyselected from 0 to 5. In some embodiments, a, g, and l are 2; b-f andi-k are 1; and h is zero. In some embodiments, a-l are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWDSGRGSYQ (SEQ ID NO: 39), a DE loop having the aminoacid sequence PGPVHT (SEQ ID NO: 41), and an FG loop having the aminoacid sequence TDHKPHADGPHTYHESP (SEQ ID NO: 43).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(m)VAGAEDYQX_(n) (SEQ ID NO:34), a DE loop having the amino acid sequence X_(o)HDLVX_(p) (SEQ ID NO:36), and an FG loop having the amino acid sequence X_(q)DMMHVEYTEHX_(r)(SEQ ID NO: 38); wherein X is any amino acid and a, b, c, d, e, f, m, n,o, p, q, and r are integers from 0 to 5, independently. In someembodiments, a and m are 2; b-f and o-r are 1; and n is zero. In someembodiments, a-f and m-r are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWVAGAEDYQ (SEQ ID NO: 33), a DE loop having the aminoacid sequence PHDLVT (SEQ ID NO: 35), and an FG loop having the aminoacid sequence TDMMHVEYTEHP (SEQ ID NO: 37).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(s)LPGKLRYQX_(t) (SEQ ID NO:60), a DE loop having the amino acid sequence X_(u)HDLRX_(w) (SEQ ID NO:62), and an FG loop having the amino acid sequence X_(y)NMMHVEYSEYX_(z)(SEQ ID NO: 64); wherein X is any amino acid and a, b, c, d, e, f, s, t,u, w, y, and z are integers from 0 to 5, independently. In someembodiments, a and s are 2; b-f, u, w, y and z are 1; and t is zero. Insome embodiments, a-f, s-u, w, y and z are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWLPGKLRYQ (SEQ ID NO: 59), a DE loop having the aminoacid sequence PHDLRT (SEQ ID NO: 61), and an FG loop having the aminoacid sequence TNMMHVEYSEYP (SEQ ID NO: 63).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(g)HERDGSRQX_(h) (SEQ ID NO:134), a DE loop having the amino acid sequence X_(i)GGVRX_(j) (SEQ IDNO: 135), and an FG loop having the amino acid sequenceX_(k)DYFNPTTHEYIYQTTX_(l)(SEQ ID NO: 136); wherein X is any amino acidand a, b, c, d, e, f, g, h, i, j, k and l are integers from 0 to 5,independently. In some embodiments, a and g are 2; b-f and i-l are 1;and h is zero. In some embodiments, a-l are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWHERDGSRQ (SEQ ID NO: 109), a DE loop having the aminoacid sequence PGGVRT (SEQ ID NO: 110), and an FG loop having the aminoacid sequence TDYFNPTTHEYIYQTTP (SEQ ID NO: 111).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(g)WAPVDRYQX_(h) (SEQ ID NO:137), a DE loop having the amino acid sequence X_(i)RDVYX_(j) (SEQ IDNO: 138), and an FG loop having the amino acid sequenceX_(k)DYKPHADGPHTYHESX_(l)(SEQ ID NO: 139); wherein X is any amino acidand a, b, c, d, e, f, g, h, i, j, k and l are integers from 0 to 5,independently. In some embodiments, a and g are 2; b-f and i-l are 1;and h is zero. In some embodiments, a-l are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWWAPVDRYQ (SEQ ID NO: 115), a DE loop having the aminoacid sequence PRDVYT (SEQ ID NO: 116), and an FG loop having the aminoacid sequence TDYKPHADGPHTYHESP (SEQ ID NO: 117).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(g)TQGSTHYQX_(h) (SEQ ID NO:146), a DE loop having the amino acid sequence X_(i)GMVYX_(j) (SEQ IDNO: 147), and an FG loop having the amino acid sequenceX_(k)DYFDRSTHEYKYRTTX_(l) (SEQ ID NO: 148); wherein X is any amino acidand a, b, c, d, e, f, g, h, i, j, k and l are integers from 0 to 5,independently. In some embodiments, a and g are 2; b-f and i-l are 1;and h is zero. In some embodiments, a-l are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWTQGSTHYQ (SEQ ID NO: 143), a DE loop having the aminoacid sequence PGMVYT (SEQ ID NO: 144), and an FG loop having the aminoacid sequence TDYFDRSTHEYKYRTTP (SEQ ID NO: 145).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(g)YWEGLPYQX_(h) (SEQ ID NO:161), a DE loop having the amino acid sequence X_(i)RDVNX_(j) (SEQ IDNO: 162), and an FG loop having the amino acid sequenceX_(k)DWYNPDTHEYIYHTIX_(l) (SEQ ID NO: 163); wherein X is any amino acidand a, b, c, d, e, f, g, h, i, j, k and l are integers from 0 to 5,independently. In some embodiments, a and g are 2; b-f and i-l are 1;and h is zero. In some embodiments, a-l are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWYWEGLPYQ (SEQ ID NO: 158), a DE loop having the aminoacid sequence PRDVNT (SEQ ID NO: 159), and an FG loop having the aminoacid sequence TDWYNPDTHEYIYHTIP (SEQ ID NO: 160).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(g)ASNRGTYQX_(h) (SEQ ID NO:176), a DE loop having the amino acid sequence X_(i)GGVSX_(j) (SEQ IDNO: 177), and an FG loop having the amino acid sequenceX_(k)DAFNPTTHEYNYFTTX_(l) (SEQ ID NO: 178); wherein X is any amino acidand a, b, c, d, e, f, g, h, i, j, k and l are integers from 0 to 5,independently. In some embodiments, a and g are 2; b-f and i-l are 1;and h is zero. In some embodiments, a-l are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWASNRGTYQ (SEQ ID NO: 173), a DE loop having the aminoacid sequence PGGVST (SEQ ID NO: 174), and an FG loop having the aminoacid sequence TDAFNPTTHEYNYFTTP (SEQ ID NO: 175).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(g)DAPTSRYQX_(h) (SEQ ID NO:190), a DE loop having the amino acid sequence X_(i)GGLSX_(j) (SEQ IDNO: 191), and an FG loop having the amino acid sequenceX_(k)DYKPHADGPHTYHESX_(l) (SEQ ID NO: 139); wherein X is any amino acidand a, b, c, d, e, f, g, h, i, j, k and l are integers from 0 to 5,independently. In some embodiments, a and g are 2; b-f and i-l are 1;and h is zero. In some embodiments, a-l are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWDAPTSRYQ (SEQ ID NO: 188), a DE loop having the aminoacid sequence PGGLST (SEQ ID NO: 189), and an FG loop having the aminoacid sequence TDYKPHADGPHTYHESP (SEQ ID NO: 117).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence X_(a)SARLKVAX_(b) (SEQ ID NO: 46), a DE loop having theamino acid sequence X_(c)KNVYX_(d) (SEQ ID NO: 48), and an FG loophaving the amino acid sequence X_(e)RFRDYQX_(f) (SEQ ID NO: 50);covalently or non-covalently linked to an EGFR binding ¹⁰Fn3 comprisinga BC loop having the amino acid sequence X_(g)DAGAVTYQX_(h) (SEQ ID NO:203), a DE loop having the amino acid sequence X_(i)GGVRX_(j) (SEQ IDNO: 135), and an FG loop having the amino acid sequenceX_(k)DYKPHADGPHTYHEYX_(l) (SEQ ID NO: 204); wherein X is any amino acidand a, b, c, d, e, f, g, h, i, j, k and l are integers from 0 to 5,independently. In some embodiments, a and g are 2; b-f and i-l are 1;and h is zero. In some embodiments, a-l are zero.

In some embodiments, an E/I binder is an antibody-like protein dimercomprising an IGFIR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the aminoacid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the aminoacid sequence TRFRDYQP (SEQ ID NO: 49); covalently or non-covalentlylinked to an EGFR binding ¹⁰Fn3 comprising a BC loop having the aminoacid sequence SWDAGAVTYQ (SEQ ID NO: 201), a DE loop having the aminoacid sequence PGGVRT (SEQ ID NO: 110), and an FG loop having the aminoacid sequence TDYKPHADGPHTYHEYP (SEQ ID NO: 202).

In some embodiments, an E/I binder is an antibody-like protein dimercomprising a) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 23-29 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 52-55 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 77-82 of SEQ ID NO: 3; covalently or non-covalently linkedto b) an EGFR binding ¹⁰Fn3 comprising a BC, DE and FG loop as set forthin any one of SEQ ID NOs: 219-327 (see e.g., FIG. 45 wherein the BC, DEand FG loop sequences for each EGFR binding ¹⁰Fn3 are underlined). Insome embodiments, an E/I binder is an antibody-like protein dimercomprising a) an IGFIR binding ¹⁰Fn3 comprising a BC loop having theamino acid sequence set forth in amino acids 23-29 of SEQ ID NO: 3, a DEloop having the amino acid sequence set forth in amino acids 52-55 ofSEQ ID NO: 3, and an FG loop having the amino acid sequence set forth inamino acids 77-82 of SEQ ID NO: 3; covalently or non-covalently linkedto b) an EGFR binding ¹⁰Fn3 comprising a BC loop having the amino acidsequence set forth in amino acids corresponding to amino acid residues23-30 of SEQ ID NO: 1 of any one of SEQ ID NOs: 5-8, 52, 66-68, 106-108,112-114, 140-142, 155-157, 170-172, 182, 185-187, 198-200, or 219-327, aDE loop having the amino acid sequence set forth in amino acidscorresponding to amino acid residues 52-55 of SEQ ID NO: 1 of any one ofSEQ ID NOs: 5-8, 52, 66-68, 106-108, 112-114, 140-142, 155-157, 170-172,182, 185-187, 198-200, or 219-327, and an FG loop having the amino acidsequence set forth in amino acids corresponding to amino acid residues77-86 of SEQ ID NO: 15-8, 52, 66-68, 106-108, 112-114, 140-142, 155-157,170-172, 182, 185-187, 198-200, or 219-327. In some embodiments, theEGFR binding ¹⁰Fn3 of the antibody-like protein dimer comprises an aminoacid sequence at least 80, 90, 95, or 100% identical to the amino acidsequence of amino acid residues corresponding to E9 of SEQ ID NO: 1through T94 of SEQ ID NO: 1 of any one of SEQ ID NOs: 5-8, 52, 66-68,106-108, 112-114, 140-142, 155-157, 170-172, 182, 185-187, 198-200, or219-327. In some embodiments, the IGFIR binding ¹⁰Fn3 of theantibody-like protein dimer has an amino acid sequence at least 80, 90,95, 98, 99, or 100% identical to the amino acid sequence of amino acidresidues corresponding to E9 of SEQ ID NO: 1 through T94 of SEQ ID NO: 1of SEQ ID NO: 3. In some embodiments, the E/I binder comprises an aminoacid sequence at least 80, 85, 90, 95, 98, 99, or 100% identical to anyone of SEQ ID NOs: 20-31, 53-58, 87-92, 98-105, 118-133, 149-154,164-169, 179-184, 192-197, 205-210 and 211-216.

Preferably, X as defined herein is a naturally occurring amino acid.

In certain embodiments, the E binders, or the E and/or I monomers of theE/I binders described herein may contain a Ser to Cys amino acidsubstitution at a position corresponding to serine 62 or serine 91 ofSEQ ID NO: 1.

In certain aspects, the disclosure provides short peptide sequences thatmediate EGFR binding. Examples of such sequences include the amino acidresidues that correspond to the BC, DE, and FG loops from SEQ ID NOs: 5,7, 82, 106, 112, 141, 156, 171, 186 and 199. Other examples of suchsequences include the amino acid residues that correspond to the BC, DE,and FG loops from SEQ ID NOs: 219-327. In some embodiments, the peptidesbind to their respective ligand with a dissociation constant (K_(D)) ofless than 500 nM, 100 nM, 50 nM, 5 nM or less. Such sequences maymediate ligand binding in an isolated form or when inserted into aparticular protein structure, such as an immunoglobulin orimmunoglobulin-like domain.

In one embodiment, an antibody-like protein dimer comprises apolypeptide having the structure A-B-C, wherein A is a polypeptidecomprising, consisting essentially of, or consisting of a ¹⁰Fn3 domainthat binds to EGFR, B is a polypeptide linker, and C is a polypeptidecomprising, consisting essentially of, or consisting of a ¹⁰Fn3 domainthat binds to IGF-IR. In another embodiment, a antibody-like proteindimer comprises a polypeptide having the structure A-B-C, wherein A is apolypeptide comprising, consisting essentially of, or consisting of a¹⁰Fn3 domain that binds to IGF-IR, B is a polypeptide linker, and C is apolypeptide comprising, consisting essentially of, or consisting of a¹⁰Fn3 domain that binds to EGFR. Specific examples of antibody-likeprotein dimers having the structure A-B-C are polypeptides comprising(i) a polypeptide having an amino acid sequence set forth in any one ofSEQ ID NOs: 20-31, 53-58, 87-92, 98-105, 118-133, 149-154, 164-169,179-184, 192-197, 205-210 and 211-216, or (ii) a polypeptide comprisingan amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99%identical to any one of the amino acid sequences set forth in SEQ IDNOs: 20-31, 53-58, 87-92, 98-105, 118-133, 149-154, 164-169, 179-184,192-197, 205-210 and 211-216.

In certain embodiments, the A or C region is a polypeptide comprising a¹⁰Fn3 domain that binds to EGFR; wherein the ¹⁰Fn3 domain has thestructure from N-terminus to C-terminus: beta strand A, loop AB, betastrand B, loop BC, beta strand C, loop CD, beta strand D, loop DE, betastrand E, loop EF, beta strand F, loop FG, beta strand G; wherein: (i)the BC loop has the amino acid sequence of SEQ ID NO: 33 or 34, the DEloop has the amino acid sequence of SEQ ID NO: 35 or 36, and the FG loophas the amino acid sequence of SEQ ID NO: 37 or 38, (ii) the BC loop hasthe amino acid sequence of SEQ ID NO: 39 or 40, the DE loop has theamino acid sequence of SEQ ID NO: 41 or 42, and the FG loop has theamino acid sequence of SEQ ID NO: 43 or 44, (iii) the BC loop has theamino acid sequence of SEQ ID NO: 59 or 60, the DE loop has the aminoacid sequence of SEQ ID NO: 61 or 62, and the FG loop has the amino acidsequence of SEQ ID NO: 63 or 64, (iv) the BC loop has the amino acidsequence of SEQ ID NO: 109 or 134, the DE loop has the amino acidsequence of SEQ ID NO: 110 or 135, and the FG loop has the amino acidsequence of SEQ ID NO: 111 or 136, (v) the BC loop has the amino acidsequence of SEQ ID NO: 115 or 137, the DE loop has the amino acidsequence of SEQ ID NO: 116 or 138, and the FG loop has the amino acidsequence of SEQ ID NO: 117 or 139, (vi) the BC loop has the amino acidsequence of SEQ ID NO: 143 or 146, the DE loop has the amino acidsequence of SEQ ID NO: 144 or 147, and the FG loop has the amino acidsequence of SEQ ID NO: 145 or 148, (vii) the BC loop has the amino acidsequence of SEQ ID NO: 158 or 161, the DE loop has the amino acidsequence of SEQ ID NO: 159 or 162, and the FG loop has the amino acidsequence of SEQ ID NO: 160 or 163, (viii) the BC loop has the amino acidsequence of SEQ ID NO: 173 or 176, the DE loop has the amino acidsequence of SEQ ID NO: 174 or 177, and the FG loop has the amino acidsequence of SEQ ID NO: 175 or 178, (ix) the BC loop has the amino acidsequence of SEQ ID NO: 188 or 190, the DE loop has the amino acidsequence of SEQ ID NO: 189 or 191, and the FG loop has the amino acidsequence of SEQ ID NO: 117 or 139, (x) the BC loop has the amino acidsequence of SEQ ID NO: 201 or 203, the DE loop has the amino acidsequence of SEQ ID NO: 110 or 135, and the FG loop has the amino acidsequence of SEQ ID NO: 202 or 204, or (xi) the BC, DE and FG loops havethe amino acid sequences as set forth in any one of SEQ ID NOs: 219-327(see e.g., FIG. 45 wherein the BC, DE and FG loops for each of SEQ IDNOs: 219-327 are underlined); wherein the ¹⁰Fn3 domain folds into anantibody heavy chain variable region-like structure; and wherein thepolypeptide binds to EGFR with a K_(D) of less than 100 nM. The ¹⁰Fn3domain that binds to EGFR preferably folds into a structure wherein the7 beta strands are distributed between two beta sheets that pack againsteach other forming a stable core and wherein the beta strands areconnected by the six loops which are solvent exposed. In exemplaryembodiments, the ¹⁰Fn3 domain is from 80-150 amino acids in length.

In exemplary embodiments, the A or C region is a ¹⁰Fn3 domain that bindsto EGFR with a K_(D) of less than 100 nM having a sequence selected fromthe group consisting of SEQ ID NO: 83-85 and 466-472 as set forth below:

(SEQ ID NO: 83) EVVAATX_(n1) SLLIX_(a1) SWVAGAEDYQX_(a2) YYRITYGEX_(n2)QEFTVX_(a3) PHDL VTX_(a4) ATIX_(n3) DYTITVYAVX_(n5) TDMMHVEYTEHPX_(n6)ISINYRT; (SEQ ID NO: 84) EVVAATX_(n1) SLLIX_(a1) SWDSGRGSYQX_(a2)YYRITYGEX_(n2) QEFTVX_(a3) PGPV HTX_(a4) ATIX_(n3) DYTITVYAVX_(a5)TDHKPHADGPHTYHESPX_(a6) ISINYRT; or (SEQ ID NO: 85) EVVAATX_(n1)SLLIX_(a1) SWLPGKLRYQX_(a2) YYRITYGEX_(n2) QEFTVX_(a3) PHDL RTX_(a4)ATIX_(n3) DYTITVYAVX_(a5) TNMMHVEYSEYPX_(a6) ISINYRT. (SEQ ID NO: 466)EVVAATX_(n1) SLLIX_(a1) SWHERDGSRQX_(a2) YYRITYGEX_(n2) QEFTVX_(a3) PGGVRTX_(a4) ATIX_(n3) DYTITVYAVX_(a5) TDYFNPTTHEYIYQTTPX_(a6) ISINYRT.(SEQ ID NO: 467) EVVAATX_(n1) SLLIX_(a1) SWWAPVDRYQX_(a2) YYRITYGEX_(n2)QEFTVX_(a3) PRDV YTX_(a4) ATIX_(n3) DYTITVYAVX_(a5)TDYKPHADGPHTYHESPX_(a6) ISINYRT. (SEQ ID NO: 468) EVVAATX_(n1)SLLIX_(a1) SWTQGSTHYQX_(a2) YYRITYGEX_(n2) QEFTVX_(a3) PGMV YTX_(a4)ATIX_(n3) DYTITVYAVX_(a5) TDYFDRSTHEYKYRTTPX_(a6) ISINYRT.(SEQ ID NO: 469) EVVAATX_(n1) SLLIX_(a1) SWYWEGLPYQX_(a2) YYRITYGEX_(n2)QEFTVX_(a3) PRDV NTX_(a4) ATIX_(n3) DYTITVYAVX_(a5)TDWYNPDTHEYIYHTIPX_(a6) ISINYRT. (SEQ ID NO: 470) EVVAATX_(n1)SLLIX_(a1) SWASNRGTYQX_(a2) YYRITYGEX_(n2) QEFTVX_(a3) PGGV STX_(a4)ATIX_(n3) DYTITVYAVX_(a5) TDAFNPTTHEYNYFTTPX_(a6) ISINYRT.(SEQ ID NO: 471) EVVAATX_(n1) SLLIX_(a1) SWDAPTSRYQX_(a2) YYRITYGEX_(n2)QEFTVX_(a3) PGGL STX_(a4) ATIX_(n3) DYTITVYAVX_(a5)TDYKPHADGPHTYHESPX_(a6) ISINYRT. (SEQ ID NO: 472) EVVAATX_(n1)SLLIX_(a1) SWDAGAVTYQX_(a2) YYRITYGEX_(n2) QEFTVX_(a3) PGGV RTX_(a4)ATIX_(n3) DYTITVYAVX_(a5) TDYKPHADGPHTYHEYPX_(a6) ISINYRT.In SEQ ID NOs: 83-85 and 466-472, the BC, DE and FG loops have a fixedsequence as shown in bold, or a sequence at least 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% identical to the sequences shown in bold, the ABloop is represented by X_(n1), the CD is represented by X_(n2), and EFloop is represented by X_(n3), and the beta strands A-G are underlined.X represents any amino acid and the subscript following the X representsan integer of the number of amino acids. In particular, n1 may beanywhere from 1-15, 2-15, 1-10, 2-10, 1-8, 2-8, 1-5, 2-5, 1-4, 2-4, 1-3,2-3, or 1-2 amino acids; n2 and n3 may each independently be anywherefrom 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10,6-8, 2-7, 5-7, or 6-7 amino acids; and a1-a6 may each independentlycomprise from 0-10, 0-5, 1-10, 1-5, or 2-5 amino acids. In preferredembodiments, n1 is 2 amino acids, n2 is 7 amino acids, n3 is 7 aminoacids, and a1-a6 is 0 amino acids. The sequences of the beta strands mayhave anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions,deletions or additions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 1. In an exemplaryembodiment, the sequences of the beta strands may have anywhere from 0to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 conservative substitutions across all 7scaffold regions relative to the corresponding amino acids shown in SEQID NO: 1. In certain embodiments, the core amino acid residues are fixedand any substitutions, conservative substitutions, deletions oradditions occur at residues other than the core amino acid residues. Incertain embodiments, the EGFR binder is represented by one of thefollowing amino acid sequences:

(SEQ ID NO: 66) EVVAATPTSLLISW VAGAEDYQ YYRITYGETGGNSPVQEFTVP HDLVTATISGLKPGVDYTITVYAVTD MMHVEYTEH PISINYRT; (SEQ ID NO: 67)EVVAATPTSLLISW DSGRGSYQ YYRITYGETGGNSPVQEFTVP GPVH TATISGLKPGVDYTITVYAVTDHKPHADGPHTYHES PISINYRT; (SEQ ID NO: 68) EVVAATPTSLLISW LPGKLRYQYYRITYGETGGNSPVQEFTVP HDLR TATISGLKPGVDYTITVYAVT NMMHVEYSEY PISINYRT;(SEQ ID NO: 108) EVVAATPTSLLISW HERDGSRQ YYRITYGETGGNSPVQEFTVP GGVRTATISGLKPGVDYTITVYAVT DYFNPTTHEYIYQTT PISINYRT; or (SEQ ID NO: 114)EVVAATPTSLLISW WAPVDRYQ YYRITYGETGGNSPVQEFTVP RDVY TATISGLKPGVDYTITVYAVTDYKPHADGPHTYHES PISINYRT. (SEQ ID NO: 141) EVVAATPTSLLISW TQGSTHYQYYRITYGETGGNSPVQEFTVP GMVY TATISGLKPGVDYTITVYAVT DYFDRSTHEYKYRTTPISINYRT (SEQ ID NO: 156) EVVAATPTSLLISW YWEGLPYQYYRITYGETGGNSPVQEFTVPRDVN TATISGLKPGVDYTITVYAVT DWYNPDTHEYIYHTI PISINYRT(SEQ ID NO: 171) EVVAATPTSLLISW ASNRGTYQ YYRITYGETGGNSPVQEFTVP GGVSTATISGLKPGVDYTITVYAVT DAFNPTTHEYNYFTT PISINYRT (SEQ ID NO: 186)EVVAATPTSLLISW DAPTSRYQ YYRITYGETGGNSPVQEFTVP GGLS TATISGLKPGVDYTITVYAVTDYKPHADGPHTYHES PISINYRT E112 (SEQ ID NO: 199) EVVAATPTSLLISW DAGAVTYQYYRITYGETGGNSPVQEFTVP GGVR TATISGLKPGVDYTITVYAVT DYKPHADGPHTYHEYPISINYRTIn SEQ ID NOs: 66-68, 108, 114, 141, 156, 171, 186 and 199, the sequenceof the BC, DE and FG loops have a fixed sequence as shown in bold, or asequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical tothe sequences shown in bold, and the remaining sequence which isunderlined (e.g., the sequence of the 7 beta strands and the AB, CD andEF loops) has anywhere from 0 to 20, from 0 to 15, from 0 to 10, from 0to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2,or from 0 to 1 substitutions, conservative substitutions, deletions oradditions relative to the corresponding amino acids shown in SEQ ID NO:66-68, 108, 114, 141, 156, 171, 186 and 199. In certain embodiments, thecore amino acid residues are fixed and any substitutions, conservativesubstitutions, deletions or additions occur at residues other than thecore amino acid residues. The ¹⁰Fn3 domain that binds to EGFR mayoptionally comprise an N-terminal extension of from 1-20, 1-15, 1-10,1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in length. ExemplaryN-terminal extensions include (represented by the single letter aminoacid code) M, MG, G, MGVSDVPRDL (SEQ ID NO: 69), GVSDVPRDL (SEQ ID NO:70), and VSDVPRDL (SEQ ID NO: 71), or N-terminal truncations of any oneof SEQ ID NOs: 69, 70, or 71. Other suitable N-terminal extensionsinclude, for example, X_(n)SDVPRDL (SEQ ID NO: 72), X_(n)DVPRDL (SEQ IDNO: 73), X_(n)VPRDL (SEQ ID NO: 74), X_(n)PRDL (SEQ ID NO: 75), X_(n)RDL(SEQ ID NO: 76), X_(n)DL (SEQ ID NO: 77), or X_(n)L, wherein n=0, 1 or 2amino acids, wherein when n=1, X is Met or Gly, and when n=2, X isMet-Gly. The ¹⁰Fn3 domain that binds to EGFR may optionally comprise aC-terminal tail. Exemplary C-terminal tails include polypeptides thatare from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids inlength. Specific examples of C-terminal tails include EIDKPSQ (SEQ IDNO: 9), EIDKPCQ (SEQ ID NO: 10), and EIDK (SEQ ID NO: 78). In otherembodiments, suitable C-terminal tails may be a C-terminally truncatedfragment of SEQ ID NOs: 9, 10 or 78, including, for example, one of thefollowing amino acid sequences (represented by the single letter aminoacid code): E, E1, EID, EIDKP (SEQ ID NO: 79), EIDKPS (SEQ ID NO: 80),or EIDKPC (SEQ ID NO: 81). Other suitable C-terminal tails include, forexample, ES, EC, EGS, EGC, EGSGS (SEQ ID NO: 96), EGSGC (SEQ ID NO: 97),or EIEK (SEQ ID NO: 217). In certain embodiments, the ¹⁰Fn3 domain thatbinds to EGFR comprises both an N-terminal extension and a C-terminaltail. In exemplary embodiments, the A region comprises an N-terminalextension beginning with Gly or Met-Gly and a C-terminal extension thatdoes not contain a cysteine residue and the B region comprises anN-terminal extension that does not start with a Met and a C-terminalextension that comprises a cysteine residue. Specific examples of ¹⁰Fn3domains that bind to EGFR are polypeptides comprising (i) a polypeptidehaving an amino acid sequence set forth in any one of SEQ ID NOs: 5-8,52, 66-68, 82-85, 106-108, 112-114, 140-142, 155-157, 170-172, 185-187,198-200, and 219-327, or (ii) a polypeptide comprising an amino acidsequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to the aminoacid sequence set forth in any one of SEQ ID NOs: 5-8, 52, 66-68, 82-85,106-108, 112-114, 140-142, 155-157, 170-172, 185-187, 198-200, and219-327.

In certain embodiments, the A or C region is a polypeptide comprising a¹⁰Fn3 domain that binds to IGF-IR, wherein the ¹⁰Fn3 domain has thestructure from N-terminus to C-terminus: beta strand A, loop AB, betastrand B, loop BC, beta strand C, loop CD, beta strand D, loop DE, betastrand E, loop EF, beta strand F, loop FG, beta strand G, wherein the BCloop has the amino acid sequence of SEQ ID NO: 45 or 46, the DE loop hasthe amino acid sequence of SEQ ID NO: 47 or 48, and the FG loop has theamino acid sequence of SEQ ID NO: 49 or 50, wherein the ¹⁰Fn3 domainfolds into an antibody heavy chain variable region-like structure, andwherein the polypeptide binds to IGF-IR with a K_(D) of less than 100nM. The ¹⁰Fn3 domain that binds to IGF-IR preferably folds into astructure wherein the 7 beta strands are distributed between two betasheets that pack against each other forming a stable core and whereinthe beta strands are connected by the six loops which are solventexposed. In exemplary embodiments, the ¹⁰Fn3 domain is from 80-150 aminoacids in length.

In exemplary embodiments, the A or C region is a ¹⁰Fn3 domain that bindsto IGF-IR with a K_(D) of less than 100 nM having the sequence set forthbelow:

(SEQ ID NO: 86) EVVAATX_(n1) SLLIX_(a1) SWSARLKVARX_(a2) YYRITYGEX_(n2)QEFTVX_(a3) PKN VYTX_(a4) ATIX_(n3) DYTITVYAVX_(a5) TRFRDYQPX_(a6)ISINYRT.In SEQ ID NO: 86, the BC, DE and FG loops have a fixed sequence as shownin bold, or a sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% identical to the sequences shown in bold, the AB loop is representedby X_(n1), the CD loop is represented by X_(n2), and the EF loop isrepresented by X_(n3), and the beta strands A-G are underlined. Xrepresents any amino acid and the subscript following the X representsan integer of the number of amino acids. In particular, n1 may beanywhere from 1-15, 2-15, 1-10, 2-10, 1-8, 2-8, 1-5, 2-5, 1-4, 2-4, 1-3,2-3, or 1-2 amino acids; n2 and n3 may each independently be anywherefrom 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10,6-8, 2-7, 5-7, or 6-7 amino acids; and a1-a6 may each independentlycomprise from 0-10, 0-5, 1-10, 1-5, or 2-5 amino acids. In preferredembodiments, n1 is 2 amino acids, n2 is 7 amino acids, n3 is 7 aminoacids, and a1-a6 is 0 amino acids. The sequences of the beta strands mayhave anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions,deletions or additions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 1. In an exemplaryembodiment, the sequences of the beta strands may have anywhere from 0to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 conservative substitutions across all 7scaffold regions relative to the corresponding amino acids shown in SEQID NO: 1. In certain embodiments, the core amino acid residues are fixedand any substitutions, conservative substitutions, deletions oradditions occur at residues other than the core amino acid residues. Incertain embodiments, the IGF-IR binder is represented by the followingamino acid sequence:

(SEQ ID NO: 65) EVVAATPTSLLISW SARLKVA RYYRITYGETGGNSPVQEFTVP KNVYTATISGLKPGVDYTITVYAVT RFRDY QPISINYRT.In SEQ ID NO: 65, the sequence of the BC, DE and FG loops have a fixedsequence as shown in bold, or a sequence at least 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% identical to the sequences shown in bold, and theremaining sequence which is underlined (e.g., the sequence of the 7 betastrands and the AB, CD and EF loops) has anywhere from 0 to 20, from 0to 15, from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4,from 0 to 3, from 0 to 2, or from 0 to 1 substitutions, conservativesubstitutions, deletions or additions relative to the correspondingamino acids shown in SEQ ID NO: 65. In certain embodiments, the coreamino acid residues are fixed and any substitutions, conservativesubstitutions, deletions or additions occur at residues other than thecore amino acid residues. The ¹⁰Fn3 domain that binds to IGF-IR mayoptionally comprise an N-terminal extension of from 1-20, 1-15, 1-10,1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in length. ExemplaryN-terminal extensions include (represented by the single letter aminoacid code) M, MG, G, MGVSDVPRDL (SEQ ID NO: 69), GVSDVPRDL (SEQ ID NO:70), and VSDVPRDL (SEQ ID NO: 71), or N-terminal truncations of any oneof SEQ ID NOs: 69, 70, or 71. Other suitable N-terminal extensionsinclude, for example, X_(n)SDVPRDL (SEQ ID NO: 72), X_(n)DVPRDL (SEQ IDNO: 73), X_(n)VPRDL (SEQ ID NO: 74), X_(n)PRDL (SEQ ID NO: 75), X_(n)RDL(SEQ ID NO: 76), X_(n)DL (SEQ ID NO: 77), or X_(n)L, wherein n=0, 1 or 2amino acids, wherein when n=1, X is Met or Gly, and when n=2, X isMet-Gly. The ¹⁰Fn3 domain that binds to IGF-IR may optionally comprise aC-terminal tail. Exemplary C-terminal tails include polypeptides thatare from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids inlength. Specific examples of C-terminal tails include EIDKPSQ (SEQ IDNO: 9), EIDKPCQ (SEQ ID NO: 10), and EIDK (SEQ ID NO: 78). In otherembodiments, suitable C-terminal tails may be a C-terminally truncatedfragment of SEQ ID NOs: 9, 10 or 78, including, for example, one of thefollowing amino acid sequences (represented by the single letter aminoacid code): E, E1, EID, EIDKP (SEQ ID NO: 79), EIDKPS (SEQ ID NO: 80),or EIDKPC (SEQ ID NO: 81). Other suitable C-terminal tails include, forexample, ES, EC, EGS, EGC, EGSGS (SEQ ID NO: 96), EGSGC (SEQ ID NO: 97),or EIEK (SEQ ID NO: 217). In certain embodiments, the 10Fn3 domain thatbinds to IGF-IR comprises both an N-terminal extension and a C-terminaltail. In exemplary embodiments, the A region comprises an N-terminalextension beginning with Gly or Met-Gly and a C-terminal extension thatdoes not contain a cysteine residue and the B region comprises anN-terminal extension that does not start with a Met and a C-terminalextension that comprises a cysteine residue. Specific examples of ¹⁰Fn3domains that bind to IGF-IR are polypeptides comprising (i) apolypeptide having an amino acid sequence set forth in any one of SEQ IDNOs: 3, 4, 65 or 86, or (ii) a polypeptide comprising an amino acidsequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to the aminoacid sequence set forth in any one of SEQ ID NOs: 3, 4, 65 or 86.

The B region is a linker as described further herein. In exemplaryembodiments, the B region is a polypeptide linker. Exemplary polypeptidelinkers include polypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5,1-4, 1-3, or 1-2 amino acids. Specific examples of suitable polypeptidelinkers are described further herein and include, for example, linkershaving a sequence selected from the group consisting of SEQ ID NOs:11-19, 51, 93-95 and 218. In certain embodiments, the linker may be aC-terminal tail polypeptide as described herein, an N-terminal extensionpolypeptide as described herein, or a combination thereof.

In one embodiment, an antibody-like protein dimer comprises apolypeptide having the structure X₁-A-X₂-B-X₃-C-X₄, wherein X₁ is anoptional N-terminal extension, A is a ¹⁰Fn3 domain that binds to EGFR,X₂ is an optional C-terminal tail, B is a polypeptide linker, X₃ is anoptional N-terminal extension, C is a ¹⁰Fn3 domain that binds to IGF-IR,and X₄ is an optional C-terminal tail. In another embodiment, anantibody-like protein dimer comprises a polypeptide having the structureX₁-A-X₂-B-X₃-C—X₄, wherein X₁ is an optional N-terminal extension, A isa ¹⁰Fn3 domain that binds to IGF-IR, X₂ is an optional C-terminal tail,B is a polypeptide linker, X₃ is an optional N-terminal extension, C isa ¹⁰Fn3 domain that binds to EGFR, and X₄ is an optional C-terminaltail. Specific examples of suitable N-terminal extensions and C-terminaltails are described above. In certain embodiments, one or more of X₁,X₂, B, X₃ or X₄ may comprise an amino acid residue suitable forpegylation, such as a cysteine or lysine residue. In exemplaryembodiments, X₄ comprises at least one amino acid suitable forpegylation, such as a cysteine or lysine residue. Specific examples ofsuitable polypeptide linkers are described further below. Specificexamples of antibody-like protein dimers having the structureX₁-A-X₂-B-X₃-C-X₄ are polypeptides comprising (i) a polypeptide havingthe amino acid sequence set forth in any one of SEQ ID NOs: 20-31,53-58, 87-92, 98-105, 118-133, 149-154, 164-169, 179-184, 192-197,205-210 and 211-216, or (ii) a polypeptide comprising an amino acidsequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to the aminoacid sequence set forth in any one of SEQ ID NOs: 20-31, 53-58, 87-92,98-105, 118-133, 149-154, 164-169, 179-184, 192-197, 205-210 and211-216.

In certain embodiments, it may be desirable to tune the potency of one¹⁰Fn3 binding domain relative to the other ¹⁰Fn3 binding domain in theantibody-like protein dimers described herein. For example, if thebinding affinity of the first ¹⁰Fn3 domain is significantly higher thanthe binding affinity of the second ¹⁰Fn3 domain, the biological effectof the first ¹⁰Fn3 domain could overwhelm the effects of the second ofsecond ¹⁰Fn3 domain. Accordingly, in certain embodiments, it may bedesirable for the binding affinities of the first and second ¹⁰Fn3domains of an antibody-like protein dimer to be similar to each other,e.g., binding affinities within 100-fold, 30-fold, 10-fold, 3-fold,1-fold, 0.3-fold or 0.1-fold, of each other, or binding affinitieswithin 0.1-fold to 10-fold, within 0.3-fold to 10-fold, within 0.1-foldto 3-fold, within 0.3-fold to 3-fold, within 0.1-fold to 1-fold, within0.3-fold to 1-fold, within 1-fold to 10-fold, within 3-fold to 10-fold,within 3-fold to 30-fold, or within 1-fold to 3-fold of each other.

Conjugation

Multimers of antibody-like proteins may be covalently or non-covalentlylinked. In some embodiments, an EGFR binding ¹⁰Fn3 may be directly orindirectly linked to an IGFIR binding ¹⁰Fn3 via a polypeptide linker.Suitable linkers for joining Fn3 are those which allow the separatedomains to fold independently of each other forming a three dimensionalstructure that permits high affinity binding to a target molecule.

The disclosure provides a number of suitable linkers that meet theserequirements, including glycine-serine based linkers, glycine-prolinebased linkers, as well as the linker having the amino acid sequencePSTSTST (SEQ ID NO: 12). The Examples described herein demonstrate thatFn3 domains joined via polypeptide linkers retain their target bindingfunction. In some embodiments, the linker is a glycine-serine basedlinker. These linkers comprise glycine and serine residues and may bebetween 8 and 50, 10 and 30, and 10 and 20 amino acids in length.Examples include linkers having an amino acid sequenceGSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 11), GSGSGSGSGS (SEQ ID NO: 13), GGGGSGGGGS GGGGS (SEQ ID NO: 14), GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 15),GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 16), or GGGGSGGGGSGGGSG (SEQID NO: 17). In some embodiments, the linker is a glycine-proline basedlinker. These linkers comprise glycine and proline residues and may bebetween 3 and 30, 10 and 30, and 3 and 20 amino acids in length.Examples include linkers having an amino acid sequence GPGPGPG (SEQ IDNO: 18), GPGPGPGPGPG (SEQ ID NO: 19), and GPG (SEQ ID NO: 51). In someembodiments, the linker is a proline-alanine based linker. These linkerscomprise proline and alanine residues and may be between 3 and 30, 10and 30, 3 and 20 and 6 and 18 amino acids in length. Examples of suchlinkers include SEQ ID NOs: 93, 94 and 95. It is contemplated, that theoptimal linker length and amino acid composition may be determined byroutine experimentation by methods well known in the art.

In some embodiments, multimers of antibody-like proteins are linked viaa polypeptide linker having a protease site that is cleavable by aprotease in the blood or target tissue. Such embodiments can be used torelease two or more therapeutic proteins for better delivery ortherapeutic properties or more efficient production compared toseparately producing such proteins.

Additional linkers or spacers, e.g., SEQ ID NOs: 9 and 10, may beintroduced at the C-terminus of a Fn3 domain between the Fn3 domain andthe polypeptide linker. Additional linkers or spacers may be introducedat the N-terminus of a Fn3 domain between the Fn3 domain and thepolypeptide linker.

In some embodiments, multimers of antibody-like proteins may be directlyor indirectly linked via a polymeric linker. Polymeric linkers can beused to optimally vary the distance between each protein moiety tocreate a protein with one or more of the following characteristics: 1)reduced or increased steric hindrance of binding of one or more proteindomain when binding to a protein of interest, 2) increased proteinstability or solubility, 3) decreased protein aggregation, and 4)increased overall avidity or affinity of the protein.

In some embodiments, multimers of antibody-like proteins are linked viaa biocompatible polymer such as a polymeric sugar. The polymeric sugarcan include an enzymatic cleavage site that is cleavable by an enzyme inthe blood or target tissue. Such embodiments can be used to release twoor more therapeutic proteins for better delivery or therapeuticproperties or more efficient production compared to separately producingsuch proteins

In some embodiments, multimers of antibody-like proteins are linked viaa polyoxyalkylene, in particular a polyethylene glycol (PEG) moiety.Antibody-like proteins may comprise a cysteine containing linker, suchas the linker set forth in SEQ ID NO: 10, 81, 97 or 218. PEG may beconjugated to the cysteine moiety in the linker sequence and mayoperably link the two domains.

Pharmacokinetic Moieties

In one aspect, the disclosure provides E binders and E/I binders furthercomprising a pharmacokinetic (PK) moiety. In some embodiments, the E/Ibinder is a multimer of antibody-like proteins, in particular, a dimerof an EGFR binding ¹⁰Fn3 and an IGFIR binding ¹⁰Fn3. Improvedpharmacokinetics may be assessed according to the perceived therapeuticneed. Often it is desirable to increase bioavailability and/or increasethe time between doses, possibly by increasing the time that a proteinremains available in the serum after dosing. In some instances, it isdesirable to improve the continuity of the serum concentration of theprotein over time (e.g., decrease the difference in serum concentrationof the protein shortly after administration and shortly before the nextadministration). E binders and E/I binders may be attached to a moietythat reduces the clearance rate of the polypeptide in a mammal (e.g.,mouse, rat, or human) by greater than three-fold relative to theunmodified polypeptide. Other measures of improved pharmacokinetics mayinclude serum half-life, which is often divided into an alpha phase anda beta phase. Either or both phases may be improved significantly byaddition of an appropriate moiety.

Moieties that tend to slow clearance of a protein from the blood includepolyoxyalkylene moieties (e.g., polyethylene glycol); sugars (e.g.,sialic acid); and well-tolerated protein moieties (e.g., Fc, Fcfragments, transferrin, or serum albumin).

In some embodiments, the PK moiety is a serum albumin binding proteinsuch as those described in U.S. Publication Nos. 2007/0178082 and2007/0269422.

In some embodiments, the PK moiety is a serum immunoglobulin bindingprotein such as those described in U.S. Publication No. 2007/0178082.

In some embodiments, the PK moiety is polyethylene glycol (PEG).

The serum clearance rate of a PK-modified antibody-like protein multimermay be decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, oreven 90%, relative to the clearance rate of the unmodified E/I binders.The PK-modified multimer may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified multimer. Thehalf-life of PK-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified multimer. In some embodiments, the multimer half-life isdetermined in vitro, such as in a buffered saline solution or in serum.In other embodiments, the multimer half-life is an in vivo half life,such as the half-life of the multimer in the serum or other bodily fluidof an animal.

In some embodiments, a PK moiety is linked to an antibody-like proteinmultimer via at least one disulfide bond, a peptide bond, a polypeptide,a polymeric sugar, or a polyethylene glycol moiety. Exemplarypolypeptide linkers include PSTSTST (SEQ ID NO: 12), EIDKPSQ (SEQ ID NO:9), and GS linkers, such as GSGSGSGSGS (SEQ ID NO: 13) and multimersthereof.

Binding/Screening

The disclosure provides E binders and E/I binders, in particular,antibody-like protein multimers such as a dimer of an EGFR binding ¹⁰Fn3and an IGFIR binding ¹⁰Fn3. Binding to EGFR or IGFIR may be assessed interms of equilibrium constants (e.g., dissociation, K_(D)) and in termsof kinetic constants (e.g., on rate constant, k_(on) and off rateconstant, k_(off)). In some embodiments, an antibody-like proteinmonomer or multimer will bind to EGFR with a K_(D) of less than 500 nM,100 nM, 50 nM, 5 nM or less. In some embodiments, an antibody-likeprotein multimer will bind to IGFIR with a K_(D) of less than 500 nM,100 nM, 50 nM, 5 nM or less. Higher K_(D) values may be tolerated wherethe k_(off) is sufficiently low or the k_(on) is sufficiently high.

E binders and E/I binders may bind to any part of EGFR, including theextracellular domain of a EGFR, in particular the ligand binding domainof EGFR. Binding of E binders and E/I binders to EGFR may disrupt theinteraction of EGFR with one or more ligands, including TGF-alpha andEGF, and/or disrupt receptor dimerization. In some embodiments, Ebinders and E/I binders compete with an anti-EGFR antibody for bindingto EGFR. The anti-EGFR antibody may be selected from any known anti-EGFRantibody including panitumumab (Amgen), nimotuzumab (YM Biosciences),zalutumumab (Genmab), EMD72000 (Merck KGaA), and cetuximab (ImCloneSystems).

In some embodiments, E binders and E/I binders inhibit downstreamsignaling of EGFR. EGFR ligand binding leads to homo- or heterodimericreceptor dimerization with EGFR or another HER family member.Dimerization promotes receptor autophosphorylation, which in turn leadsto the activation of several signaling pathways.

E/I binders may bind to any part of IGFIR, including the extracellulardomain of a IGFIR, in particular the ligand binding domain of IGFIR.Binding of E/I binders to IGFIR may disrupt the interaction of IGFIRwith one or more ligands, e.g., IGF-I and IGF-II; and/or disruptassembly of receptor heterotetramers. In some embodiments, E/I binderscompete with an anti-IGFIR antibody for binding to IGFIR. The anti-IGFIRantibody may be selected from any known anti-IGFIR antibody.

In some embodiments, E/I binders inhibit downstream signaling of IGFIR.The IGF-I receptor is composed of two types of subunits: an alphasubunit (a 130-135 kDa protein that is entirely extracellular andfunctions in ligand binding) and a beta subunit (a 95-kDa transmembraneprotein, with transmembrane and cytoplasmic domains). IGFIR is initiallysynthesized as a single chain proreceptor polypeptide that is processedby glycosylation, proteolytic cleavage, and covalent bonding to assembleinto a mature 460-kDa heterotetramer comprising two alpha-subunits andtwo beta-subunits. The beta subunit(s) possesses ligand-activatedtyrosine kinase activity.

EGFR and IGFIR receptor signaling independently activates the MAPKpathway, including the phosphorylation of MEK. Another activated pathwayis the phosphatidylinositol 3-kinase (PI3K) pathway, includingphosphorylation of AKT. Receptor signaling is transduced to the nucleus,resulting in the activation of various transcription factors.

Screening assays may be designed to identify and characterize E bindersand E/I binders. Binding assays, such as surface plasmon resonance andELISA, and assays that detect activated signaling pathways arewell-known in the art, see e.g., Example 5. Various antibodies,including many that are commercially available, have been produced whichspecifically bind to phosphorylated, activated isoforms of EGFR andIGFIR, see e.g., Examples 6 and 7. Downstream signaling events may alsobe used as an indicator of receptor inhibition, such as by measuringlevels of AKT phosphorylation, see e.g., Example 8. Cell proliferationassays are also a useful method for characterizing the ability ofcandidate E/I binders to bind and inhibit EGFR and IGFIR signaling, seee.g., Example 9.

Polymer Conjugation

Conjugation to a biocompatible polymer may be used to link antibody-likeprotein multimers and/or to improve the pharmacokinetics of theproteins. The identity, size and structure of the polymer is selected soas to improve the circulation half-life of the multimer or decrease theantigenicity of the multimer without an unacceptable decrease inactivity.

Examples of polymers useful in the invention include, but are notlimited to, poly(alkylene glycols) such as polyethylene glycol (PEG).The polymer is not limited to a particular structure and can be linear(e.g., alkoxy PEG or bifunctional PEG), or non-linear such as branched,forked, multi-armed (e.g., PEGs attached to a polyol core), anddendritic.

Typically, PEG and other water-soluble polymers (i.e., polymericreagents) are activated with a suitable activating group appropriate forcoupling to a desired site on the polypeptide. Thus, a polymeric reagentwill possess a reactive group for reaction with the polypeptide.Representative polymeric reagents and methods for conjugating thesepolymers to an active moiety are well-known in the art and furtherdescribed in Zalipsky, S., et al., “Use of Functionalized Poly(EthyleneGlycols) for Modification of Polypeptides” in Polyethylene GlycolChemistry: Biotechnical and Biomedical Applications, J. M. Harris,Plenus Press, New York (1992), and in Zalipsky (1995) Advanced DrugReviews 16: 157-182.

Typically, the weight-average molecular weight of the polymer is fromabout 100 Daltons to about 150,000 Daltons. Exemplary weight-averagemolecular weights for the biocompatible polymer include about 20,000Daltons, about 40,000 Daltons, about 60,000 Daltons and about 80,000Daltons. Branched versions of the biocompatible polymer having a totalmolecular weight of any of the foregoing can also be used.

In some embodiments, the polymer is PEG. PEG is a well-known, watersoluble polymer that is commercially available or can be prepared byring-opening polymerization of ethylene glycol according to methods wellknown in the art (Sandler and Karo, Polymer Synthesis, Academic Press,New York, Vol. 3, pages 138-161). The term “PEG” is used broadly toencompass any polyethylene glycol molecule, without regard to size or tomodification at an end of the PEG, and can be represented by theformula: X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH, where n is 20 to 2300 and X is H ora terminal modification, e.g., a C₁₋₄ alkyl. PEG can contain furtherchemical groups which are necessary for binding reactions, which resultfrom the chemical synthesis of the molecule; or which act as a spacerfor optimal distance of parts of the molecule. In addition, such a PEGcan consist of one or more PEG side-chains which are linked together.PEGs with more than one PEG chain are called multiarmed or branchedPEGs. Branched PEG are described in, for example, European PublishedApplication No. 473084A and U.S. Pat. No. 5,932,462.

To effect covalent attachment of the polymer molecule(s) to apolypeptide, the hydroxyl end groups of the polymer molecule must beprovided in activated form, i.e. with reactive functional groups.Suitably activated polymer molecules are commercially available, e.g.from Nektar Therapeutics, Inc., Huntsville, Ala., USA; PolyMASCPharmaceuticals plc, UK; or SunBio Corporation, Anyang City, SouthKorea. Alternatively, the polymer molecules can be activated byconventional methods known in the art, e.g. as disclosed in WO 90/13540.Specific examples of activated PEG polymers include the following linearPEGs: NHS-PEG, SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG,SG-PEG, SCM-PEG, NOR-PEG, BTC-PEG, EPDX-PEG, NCO-PEG, NPC-PEG, CDI-PEG,ALD-PEG, TRES-PEG, VS-PEG, OPSS-PEG, IODO-PEG, and MAL-PEG, and branchedPEGs, such as PEG2-NHS, PEG2-MAL, and those disclosed in U.S. Pat. No.5,932,462 and U.S. Pat. No. 5,643,575, both of which are incorporatedherein by reference.

In some embodiments where PEG molecules are conjugated to cysteineresidues on an antibody-like protein multimer, the cysteine residues arenative to the protein, whereas in other embodiments, one or morecysteine residues are engineered into the protein. Mutations may beintroduced into a protein coding sequence to generate cysteine residues.This might be achieved, for example, by mutating one or more amino acidresidues to cysteine. Preferred amino acids for mutating to a cysteineresidue include serine, threonine, alanine and other hydrophilicresidues. Preferably, the residue to be mutated to cysteine is asurface-exposed residue. Algorithms are well-known in the art forpredicting surface accessibility of residues based on primary sequenceor a protein. Alternatively, surface residues may be predicted bycomparing the amino acid sequences of binding polypeptides, given thatthe crystal structure of the framework based on which bindingpolypeptides are designed and evolved has been solved (see Himanen etal., Nature. (2001) 20-27; 414(6866):933-8) and thus the surface-exposedresidues identified. In some embodiments, cysteine residues areintroduced into antibody-like protein multimers at or near the N- and/orC-terminus, or within loop regions. Pegylation of cysteine residues maybe carried out using, for example, PEG-maleiminde, PEG-vinylsulfone,PEG-iodoacetamide, or PEG-orthopyridyl disulfide.

In some embodiments, the pegylated antibody-like protein multimercomprises a PEG molecule covalently attached to the alpha amino group ofthe N-terminal amino acid. Site specific N-terminal reductive aminationis described in Pepinsky et al., (2001) JPET, 297, 1059, and U.S. Pat.No. 5,824,784. The use of a PEG-aldehyde for the reductive amination ofa protein utilizing other available nucleophilic amino groups isdescribed in U.S. Pat. No. 4,002,531, in Wieder et al., (1979) J. Biol.Chem. 254, 12579, and in Chamow et al., (1994) Bioconjugate Chem. 5,133.

In another embodiment, pegylated antibody-like protein multimercomprises one or more PEG molecules covalently attached to a linker,which in turn is attached to the alpha amino group of the amino acidresidue at the N-terminus of the binding polypeptide. Such an approachis disclosed in U.S. Publication No. 2002/0044921 and PCT PublicationNo. WO 94/01451.

In some embodiments, an antibody-like protein multimer is pegylated atthe C-terminus. A protein may be pegylated at the C-terminus by theintroduction of C-terminal azido-methionine and the subsequentconjugation of a methyl-PEG-triarylphosphine compound via the Staudingerreaction. This C-terminal conjugation method is described in Cazalis etal., C-Terminal Site-Specific PEGylation of a Truncated ThrombomodulinMutant with Retention of Full Bioactivity, Bioconjug Chem. 2004;15(5):1005-1009.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated antibody-like protein multimers, such assize exclusion (e.g., gel filtration) and ion exchange chromatography.Products may also be separated using SDS-PAGE. Products that may beseparated include mono-, di-, tri-, poly- and un-pegylated bindingpolypeptide, as well as free PEG. The percentage of mono-PEG conjugatescan be controlled by pooling broader fractions around the elution peakto increase the percentage of mono-PEG in the composition. About ninetypercent mono-PEG conjugates represents a good balance of yield andactivity.

In some embodiments, the pegylated antibody-like protein multimers willpreferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%or 100% of the biological activity associated with the unmodifiedprotein. In some embodiments, biological activity refers to its abilityto bind to EGFR and IGFIR, as assessed by K_(D), k_(on) or k_(off). Insome embodiments, the pegylated antibody-like protein multimer shows anincrease in binding to EGFR and/or IGFIR relative to unpegylatedprotein.

Deimmunization of Binding Polypeptides

The amino acid sequences of E binders and E/I binders, in particular,antibody-like protein multimers, such as a dimer of an EGFR binding¹⁰Fn3 and an IGFIR binding ¹⁰Fn3, may be altered to eliminate one ormore B- or T-cell epitopes. A protein, or a multimer of proteins, may bedeimmunized to render it non-immunogenic, or less immunogenic, to agiven species. Deimmunization can be achieved through structuralalterations to the protein. Any deimmunization technique known to thoseskilled in the art can be employed, see e.g., WO 00/34317, thedisclosure of which is incorporated herein in its entirety.

In one embodiment, the sequences of the E binders and E/I binders can beanalyzed for the presence of MHC class II binding motifs. For example, acomparison may be made with databases of MHC-binding motifs such as, forexample by searching the “motifs” database on the worldwide web atsitewehil.wehi.edu.au. Alternatively, MHC class II binding peptides maybe identified using computational threading methods such as thosedevised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)) wherebyconsecutive overlapping peptides from the polypeptide are testing fortheir binding energies to MHC class II proteins. Computational bindingprediction algorithms include iTope™, Tepitope, SYFPEITHI, EpiMatrix(EpiVax), and MHCpred. In order to assist the identification of MHCclass II-binding peptides, associated sequence features which relate tosuccessfully presented peptides such as amphipathicity and Rothbardmotifs, and cleavage sites for cathepsin B and other processing enzymescan be searched for.

Having identified potential (e.g. human) T-cell epitopes, these epitopesare then eliminated by alteration of one or more amino acids, asrequired to eliminate the T-cell epitope. Usually, this will involvealteration of one or more amino acids within the T-cell epitope itself.This could involve altering an amino acid adjacent the epitope in termsof the primary structure of the protein or one which is not adjacent inthe primary structure but is adjacent in the secondary structure of themolecule. The usual alteration contemplated will be amino acidsubstitution, but it is possible that in certain circumstances aminoacid addition or deletion will be appropriate. All alterations can beaccomplished by recombinant DNA technology, so that the final moleculemay be prepared by expression from a recombinant host, for example bywell established methods, but the use of protein chemistry or any othermeans of molecular alteration may also be used.

Once identified T-cell epitopes are removed, the deimmunized sequencemay be analyzed again to ensure that new T-cell epitopes have not beencreated and, if they have, the epitope(s) can be deleted.

Not all T-cell epitopes identified computationally need to be removed. Aperson skilled in the art will appreciate the significance of the“strength” or rather potential immunogenicity of particular epitopes.The various computational methods generate scores for potentialepitopes. A person skilled in the art will recognize that only the highscoring epitopes may need to be removed. A skilled person will alsorecognize that there is a balance between removing potential epitopesand maintaining binding affinity of the protein. Therefore, one strategyis to sequentially introduce substitutions into the protein and thentest for antigen binding and immunogenicity.

In one aspect, the deimmunized protein is less immunogenic (or rather,elicits a reduced HAMA response) than the original protein in a humansubject. Assays to determine immunogenicity are well within theknowledge of the skilled person. Art-recognized methods of determiningimmune response can be performed to monitor a HAMA response in aparticular subject or during clinical trials. Subjects administereddeimmunized protein can be given an immunogenicity assessment at thebeginning and throughout the administration of said therapy. The HAMAresponse is measured, for example, by detecting antibodies to thedeimmunized protein in serum samples from the subject using a methodknown to one in the art, including surface plasmon resonance technology(BIAcore) and/or solid-phase ELISA analysis. Alternatively, in vitroassays designed to measure a T-cell activation event are also indicativeof immunogenicity.

Additional Modifications

In certain embodiments, E binders and E/I binders, in particular,antibody-like protein multimers such as a dimer of an EGFR binding ¹⁰Fn3and an IGFIR binding ¹⁰Fn3, may further comprise post-translationalmodifications. Exemplary post-translational protein modification includephosphorylation, acetylation, methylation, ADP-ribosylation,ubiquitination, glycosylation, carbonylation, sumoylation, biotinylationor addition of a polypeptide side chain or of a hydrophobic group. As aresult, the modified E binders and E/I binders may contain non-aminoacid elements, such as lipids, poly- or mono-saccharide, and phosphates.A preferred form of glycosylation is sialylation, which conjugates oneor more sialic acid moieties to the polypeptide. Sialic acid moietiesimprove solubility and serum half-life while also reducing the possibleimmunogenicity of the protein. See, e.g., Raju et al. Biochemistry. 2001Jul. 31; 40(30):8868-76. Effects of such non-amino acid elements on thefunctionality of an E binder or E/I binder may be tested for itsantagonizing role in EGFR and IGFIR signaling function.

In some embodiments, E binders and E/I binders are modified to enhanceantigen-dependent cell-mediated cytotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC). In some embodiments, the E/I binder is adimer of an EGFR binding ¹⁰Fn3 and an IGFIR binding ¹⁰Fn3, furthercomprising an Fc region. In some embodiments, the Fc region is a variantthat enhances ADCC or CDC. The Fc region variant may comprise a human Fcregion sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region)comprising an amino acid modification (e.g., a substitution) at one ormore amino acid positions, including positions 256, 290, 298, 312, 326,330, 333, 334, 360, 378 or 430, wherein the numbering of the residues inthe Fc region is that of the EU index as in Kabat.

Vectors & Polynucleotides Embodiments

Also included in the present disclosure are nucleic acid sequencesencoding any of the proteins described herein. As appreciated by thoseskilled in the art, because of third base degeneracy, almost every aminoacid can be represented by more than one triplet codon in a codingnucleotide sequence. In addition, minor base pair changes may result ina conservative substitution in the amino acid sequence encoded but arenot expected to substantially alter the biological activity of the geneproduct. Therefore, a nucleic acid sequence encoding a protein describedherein may be modified slightly in sequence and yet still encode itsrespective gene product.

Exemplary nucleic acids encoding the E/I binders described hereininclude nucleic acids having SEQ ID NOs: 442-465 or nucleic acids havinga sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identicalto any one of SEQ ID NOs: 442-465. Isolated nucleic acids which differfrom the nucleic acids as set forth in SEQ ID NOs: 442-465 due todegeneracy in the genetic code are also within the scope of theinvention. Also provided are E/I binders comprising an I monomer encodedby a nucleotide sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to SEQ ID NO: 328 and/or E/I binders comprising an Emonomer encoded by a nucleotide sequence at 80%, 85%, 90%, 95%, 97%,98%, 99% or 100% identical to any one of SEQ ID NOs: 329-441 or 495.Also provided are E binders encoded by a nucleotide sequence at 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs:329-441 or 495. In certain embodiments, the nucleotide sequencesencoding the E/I binders, an E monomer, or an I monomer do not contain asequence encoding a 6×His tag (SEQ ID NO: 487).

Nucleic acids encoding any of the various proteins or polypeptidesdisclosed herein may be synthesized chemically. Codon usage may beselected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc Natl Acad Sci USA. 2003 100(2):438-42; Sinclair etal. Protein Expr Purif. 2002 (1):96-105; Connell N D. Curr OpinBiotechnol. 2001 (5):446-9; Makrides et al. Microbiol. Rev. 199660(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.

General techniques for nucleic acid manipulation are within the purviewof one skilled in the art and are also described for example in Sambrooket al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold SpringHarbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., CurrentProtocols in Molecular Biology (Green Publishing and Wiley-Interscience:New York, 1987) and periodic updates, herein incorporated by reference.The DNA encoding a protein is operably linked to suitabletranscriptional or translational regulatory elements derived frommammalian, viral, or insect genes. Such regulatory elements include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences that control the termination of transcription andtranslation. The ability to replicate in a host, usually conferred by anorigin of replication, and a selection gene to facilitate recognition oftransformants are additionally incorporated. Suitable regulatoryelements are well-known in the art.

The proteins described herein may be produced as a fusion protein with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process a native signal sequence, the signal sequenceis substituted by a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion, the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces alpha-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in PCT Publication No. WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such precursor regions may be ligated in readingframe to DNA encoding the protein.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the multivalent antibody.One useful transcription termination component is the bovine growthhormone polyadenylation region. See PCT Publication No. WO 94/11026 andthe expression vector disclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, New York,1985), the relevant disclosure of which is hereby incorporated byreference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow and Summers, (Bio/Technology, 6:47,1988). In some instance it will be desired to produce proteins invertebrate cells, such as for glycosylation, and the propagation ofvertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. For many applications, the small size of theprotein multimers described herein would make E. coli the preferredmethod for expression.

Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the proteins of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes, the nucleic acids encoding the proteins mustbe modified to allow in vitro transcription to produce mRNA and to allowcell-free translation of the mRNA in the particular cell-free systembeing utilized. Exemplary eukaryotic cell-free translation systemsinclude, for example, mammalian or yeast cell-free translation systems,and exemplary prokaryotic cell-free translation systems include, forexample, bacterial cell-free translation systems.

Proteins disclosed herein can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The proteins disclosed herein can be purified by isolation/purificationmethods for proteins generally known in the field of protein chemistry.Non-limiting examples include extraction, recrystallization, salting out(e.g., with ammonium sulfate or sodium sulfate), centrifugation,dialysis, ultrafiltration, adsorption chromatography, ion exchangechromatography, hydrophobic chromatography, normal phase chromatography,reversed-phase chromatography, gel filtration, gel permeationchromatography, affinity chromatography, electrophoresis, countercurrentdistribution or any combinations of these. After purification, proteinsmay be exchanged into different buffers and/or concentrated by any of avariety of methods known to the art, including, but not limited to,filtration and dialysis.

The purified proteins are preferably at least 85% pure, more preferablyat least 95% pure, and most preferably at least 98% pure. Regardless ofthe exact numerical value of the purity, the proteins are sufficientlypure for use as a pharmaceutical product.

Imaging, Diagnostic and Other Applications

The E binders described herein can be detectably labeled and used tocontact cells expressing EGFR for imaging or diagnostic applications.The E/I binders described herein can be detectably labeled and used tocontact cells expressing EGFR and/or IGFIR for imaging or diagnosticapplications. Any method known in the art for conjugating a protein tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

In certain embodiments, the E binders and E/I binders described hereinare further attached to a label that is able to be detected (e.g., thelabel can be a radioisotope, fluorescent compound, enzyme or enzymeco-factor). The label may be a radioactive agent, such as: radioactiveheavy metals such as iron chelates, radioactive chelates of gadoliniumor manganese, positron emitters of oxygen, nitrogen, iron, carbon, orgallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ¹²³I, ¹²⁵I, ¹³¹I, ¹³²I, or⁹⁹Tc. An E binder or E/I binder affixed to such a moiety may be used asan imaging agent and is administered in an amount effective fordiagnostic use in a mammal such as a human and the localization andaccumulation of the imaging agent is then detected. The localization andaccumulation of the imaging agent may be detected by radioscintigraphy,nuclear magnetic resonance imaging, computed tomography or positronemission tomography. As will be evident to the skilled artisan, theamount of radioisotope to be administered is dependent upon theradioisotope. Those having ordinary skill in the art can readilyformulate the amount of the imaging agent to be administered based uponthe specific activity and energy of a given radionuclide used as theactive moiety.

E binders and E/I binders also are useful as affinity purificationagents. In this process, the proteins are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art. The proteins can be employed in any known assay method, suchas competitive binding assays, direct and indirect sandwich assays, andimmunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987)).

E binders are useful in methods for detecting EGFR in a sample. E/Ibinders also are useful in methods for detecting EGFR and/or IGFIR in asample. The sample will often by a biological sample, such as a biopsy,and particularly a biopsy of a tumor, a suspected tumor. The sample maybe from a human or other mammal. The E binder or E/I binder may belabeled with a labeling moiety, such as a radioactive moiety, afluorescent moiety, a chromogenic moiety, a chemiluminescent moiety, ora hapten moiety; and may be immobilized on a solid support. Detectionmay be carried out using any technique known in the art, such as, forexample, radiography, immunological assay, fluorescence detection, massspectroscopy, or surface plasmon resonance.

Therapeutic/In Vivo Uses

The E binders described herein are also useful in methods for treatingconditions which respond to an inhibition of EGFR biological activity.The E/I binders described herein are also useful in methods for treatingconditions which respond to an inhibition of EGFR and/or IGFIRbiological activity. EGFR and IGFIR are involved either directly orindirectly in the signal transduction pathways of various cellactivities, including proliferation, adhesion and migration, as well asdifferentiation.

In one aspect, the application provides methods for treating a subjectafflicted with a hyperproliferative disorder with a therapeuticallyeffective amount of an E binder or an E/I binder. In particular, Ebinders and E/I binders are useful for the treatment and/or prophylaxisof tumors and/or tumor metastases. In exemplary embodiments, the E/Ibinder is an antibody-like protein multimer such as a dimer of an EGFRbinding ¹⁰Fn3 and an IGFIR binding ¹⁰Fn3.

In some embodiments, pharmaceutical compositions comprising E binders orE/I binders are administered to a subject afflicted with a tumor,including but not limited to, a brain tumor, tumor of the urogenitaltract, tumor of the lymphatic system, stomach tumor, laryngeal tumor,monocytic leukemia, lung adenocarcinoma, small-cell lung carcinoma,pancreatic cancer, glioblastoma and breast carcinoma; or a cancerousdisease, including but not limited to, squamous cell carcinoma, bladdercancer, stomach cancer, liver cancer, kidney cancer, colorectal cancer,breast cancer, head cancer, neck cancer, oesophageal cancer,gynecological cancer, thyroid cancer, lymphoma, chronic leukemia andacute leukemia.

An E binder or an E/I binder can be administered alone or in combinationwith one or more additional therapies such as chemotherapy radiotherapy,immunotherapy, surgical intervention, or any combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described herein. Techniquesand dosages for administration vary depending on the type of specificpolypeptide and the specific condition being treated but can be readilydetermined by the skilled artisan.

Additional Agents that May be Used with E/I Binders

One aspect of the application provides combinations of E binder or E/Ibinders and an additional therapeutic agent, such as a cytotoxic agent.In some embodiments, an E binder or E/I binder is linked to a cytotoxicagent. Such embodiments can be prepared by in vitro or in vivo methodsas appropriate. In vitro methods include conjugation chemistry well knowin the art, such as conjugation to cysteine and lysine residues. Inorder to link a cytotoxic agent to a polypeptide, a linking group orreactive group is used. Suitable linking groups are well known in theart and include disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups and esterase labile groups.Cytotoxic agents can also be linked to E binders or E/I binders throughan intermediary carrier molecule such as serum albumin

Exemplary cytotoxic agents that may be linked to E binders or E/Ibinders, include maytansinoids, taxanes, analogs of CC-1065, bacterialtoxin, plant toxin, ricin, abrin, a ribonuclease (RNase), DNase I, aprotease, Staphylococcal enterotoxin-A, pokeweed antiviral protein,gelonin, diphtherin toxin, Pseudomonas exotoxin, Pseudomonas endotoxin,Ranpimase (Rap), Rap (N69Q), methotrexate, daunorubicin, doxorubicin,vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, andcalicheamicin.

In other therapeutic treatments or compositions, E binders or E/Ibinders are co-administered, or administered sequentially, with one ormore additional therapeutic agents. Suitable therapeutic agents include,but are not limited to, cytotoxic or cytostatic agents, such as cancertherapeutic agents.

Cancer therapeutic agents are those agents that seek to kill or limitthe growth of cancer cells while having minimal effects on the patient.Thus, such agents may exploit any difference in cancer cell properties(e.g., metabolism, vascularization or cell-surface antigen presentation)from healthy host cells. Therapeutic agents that can be combined withE/I binders for improved anti-cancer efficacy include diverse agentsused in oncology practice (Reference: Cancer, Principles & Practice ofOncology, DeVita, V. T., Hellman, S., Rosenberg, S. A., 6th edition,Lippincott-Raven, Philadelphia, 2001), such as doxorubicin, epirubicin,cyclophosphamide, trastuzumab, capecitabine, tamoxifen, toremifene,letrozole, anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin,carboplatin, cisplatin, dexamethasone, antide, bevacizumab,5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide,topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone,abarelix, zoledronate, streptozocin, rituximab, idarubicin, busulfan,chlorambucil, fludarabine, imatinib, cytarabine, ibritumomab,tositumomab, interferon alpha-2b, melphalam, bortezomib, altretamine,asparaginase, gefitinib, erlonitib, anti-EGF receptor antibody (e.g.,cetuximab or panitumab), ixabepilone, epothilones or derivativesthereof, platinum agents (such as carboplatin, oxaliplatin, cisplatin),taxanes (such as paclitaxel, docetaxel), and camptothecin.

Therapeutic Formulations and Modes of Administration

The present application provides methods for treating conditions whichrespond to an inhibition of EGFR and/or IGFIR biological activity.Techniques and dosages for administration vary depending on the type ofspecific polypeptide and the specific condition being treated but can bereadily determined by the skilled artisan. In general, regulatoryagencies require that a protein reagent to be used as a therapeutic isformulated so as to have acceptably low levels of pyrogens. Accordingly,therapeutic formulations will generally be distinguished from otherformulations in that they are substantially pyrogen free, or at leastcontain no more than acceptable levels of pyrogen as determined by theappropriate regulatory agency (e.g., FDA).

In some embodiments, the E binders and E/I binders are pharmaceuticallyacceptable to a mammal, in particular a human. A “pharmaceuticallyacceptable” polypeptide refers to a polypeptide that is administered toan animal without significant adverse medical consequences. Examples ofpharmaceutically acceptable E binders and E/I binders include ¹⁰Fn3domains that lack the integrin-binding domain (RGD) and ¹⁰Fn3 domainsthat are essentially endotoxin free or have very low endotoxin levels.

Therapeutic compositions may be administered with a pharmaceuticallyacceptable diluent, carrier, or excipient, in unit dosage form.Administration may be parenteral (e.g., intravenous, subcutaneous),oral, or topical, as non-limiting examples. In addition, any genetherapy technique using nucleic acids encoding E binders or E/I binders,may be employed, such as naked DNA delivery, recombinant genes andvectors, cell-based delivery, including ex vivo manipulation ofpatients' cells, and the like.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; a liquid forintravenous, subcutaneous or parenteral administration; or a gel,lotion, ointment, cream, or a polymer or other sustained release vehiclefor local administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the E binder orE/I binder is administered at about 0.01 μg/kg to about 50 mg/kg perday, preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably0.1 mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or less frequently(e.g., once every other day, once or twice weekly, or monthly). Inaddition, as is known in the art, adjustments for age as well as thebody weight, general health, sex, diet, time of administration, druginteraction, and the severity of the disease may be necessary, and willbe ascertainable with routine experimentation by those skilled in theart.

EXEMPLIFICATION

The invention now being generally described will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Summary of Sequences

Many of the sequences referenced in this application are summarized inthe table below. Unless otherwise specified, N-terminal extensions areindicated with a single underline, C-terminal tails are indicated with adouble underline, and linker sequences are indicated in bold.

SEQ ID NO: Description Sequence   1 WT human ¹⁰Fn3 domainVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITV YAVTGRGDSPASSKPISINYRT   2Variant human ¹⁰Fn3 with the VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYintegrin binding motif removed GETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITV(RGD changed to SGE; changes YAVTGSGESPASSKPISINYRTfrom SEQ ID NO: 1 are underlined)   3 I1 IRG-IR monomer with N-terminalVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY extension (N + 8) and no tailGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV YAVTRFRDYQPISINYRT   4I1 IGF-IR monomer with N-terminal MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRIextension (N + 10) and Ser tail TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTIwith His tag TVYAVTRFRDYQPISINYRTEIDKPSQHHHHHH   5E2 EGFR monomer with N-terminal VSDVPRDLEVVAATPTSLLISWDSGRGSYQYYRITYextension (N + 8) and no tail GETGGNSPVQEFTVPGPVHTATISGLKPGVDYTITVYAVTDHKPHADGPHTYHESPISINYRT   6 E2 EGFR monomer with N-terminalMGVSGVPRDLEVVAATPTSLLISWDSGRGSYQYYRI extension (N + 10) and Ser tailTYGETGGNSPVQEFTVPGPVHTATISGLKPGVDYTI with his tagTVYAVTDHKPHADGPHTYHESPISINYRTEIDKPSQ HHHHHH   7E1 EGFR monomer with N-terminal VSDVPRDLEVVAATPTSLLISWVAGAEDYQYYRITYextension (N + 8) and no tail GETGGNSPVQEFTVPHDLVTATISGLKPGVDYTITVYAVTDMMHVEYTEHPISINYRT   8 E1 EGFR monomer with N-terminalMGVSDVPRDLEVVAATPTSLLISWVAGAEDYQYYRI extension (N + 10) and Ser tailTYGETGGNSPVQEFTVPHDLVTATISGLKPGVDYTI with his tagTVYAVTDMMHVEYTEHPISINYRTEIDKPSQHHHHH H   9 Ser tail EIDKPSQ  10 Cys tailEIDKPCQ  11 (GS)₁₀ Linker GSGSGSGSGSGSGSGSGSGS  12 Fn Based LinkerPSTSTST  13 (GS)₅ Linker GSGSGSGSGS  14 (GGGGS)₃ LinkerGGGGS GGGGS GGGGS  15 (GGGGS)₄ Linker GGGGS GGGGS GGGGS GGGGS  16(GGGGS)₅ Linker GGGGS GGGGS GGGGS GGGGS GGGGS  17 G₄SG₄SG₃SG LinkerGGGGS GGGGS GGGSG  18 Linker GPGPGPG  19 Linker GPGPGPGPGPG  20I1-GS10-E2: I/E tandem having VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYI1 (with N-terminal extension GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV (N +8) and short tail) fused via YAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSGSGS₁₀ linker (GS10 is SEQ ID NO: GSGSGS VSDVPRDLEVVAATPTSLLISWDSGRGSYQ11) to E2 (with N-terminal YYRITYGETGGNSPVQEFTVPGPVHTATISGLKGPVextension (N + 8) and no tail) DYTITVYAVTDHKPHADGPHTYHESPISINYRT  21I1-GS10-E2: I/E tandem having MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRII1 (with N-terminal extension TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI (N +10) and short tail) fused TVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSvia GS₁₀ linker (GS10 is SEQ ID GSGSGSGS VSVDPRDLEVVAATPTSLLISWDSGRGSNO: 11) to E2 (with N-terminal YQYYRITYGETGGNSPVQEFTVPGPVHTATISGLKPextension (N + 8) and Ser tail) GVDYTITVYAVTDHKPHADGPHTYHESPISINYRTEIDKPSQ  22 I1-GS10-E2: I/E tandem I1 (withMGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI N-terminal extension (N + 10)TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI and short tail) fused via GS₁₀TVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS linker (GS10 is SEQ ID NO: 11) toGSGSGSGS VSDVPRDLEVVAATPTSLLISWDSGRGS E2 (with N-terminal extensionYQYYRITYGETGGNSPVQEFTVPGPVHTATISGLKP (N + 8) and Ser tail with his tag)GVDYTITVYAVTDHKPHADGPHTYHESPISINYRTE IDKPSQHHHHHH  23E2-GS10-I1: E/I tandem having E2 VSDVPRDLEVVAATPTSLLISWDSGRGSYQYYRITY(with N-terminal extension (N + GETGGNSPVQEDTVPGPVHTATISGLKPGVDYTITV8) and short tail) fused via GS₁₀ YAVTDHKPHADGPHTYHESPISINYRTEIDK GSGSGlinker (GS10 is SEQ ID NO: 11) to SGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISI1 (with N-terminal extension WSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTA (N +8) and Ser tail) TISGLKPGVDYTITVYAVTRFRDYQPISINYRTEID KPSQ  24E2-GS10-I1: E/I tandem having E2 MGVSDVPRDLEVVAATPTSLLISWDSGRGSYQYYRI(with N-terminal extension (N + TYGETGGNSPVQEFTVPGPVHTATISGLKPGVDYTI10) and short tail) fused via GS₁₀ TVYAVTDHKPHADGPHTYHESPISINYRTEIDK GSGlinker (GS10 is SEQ ID NO: 11) to SGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLLI1 (with N-terminal extension ISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVY (N +8) and Ser tail) TATISGLKPGVDYTITVYAVTRFRDYQPISINYRTE IDKPSQ  25E2-GS10-I1: E/I tandem having E2 MGVSDVPRDLEVVAATPTSLLISWDSGRGSYQYYRI(with N-terminal extension (N + TYGETGGNSPVQEFTVPGPVIITATISGLKPGVDYT10) and short tail) fused via GS₁₀ ITVYAVTDHKPHADGPHTYHESPISINYRTEIDK GSlinker (GS10 is SEQ ID NO: 11) to GSGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLI1 (with N-terminal extension LISWSARLKVARYYRITYGETGGNSPVQEFTVPKNV (N +8) and Ser tail with his tag) YTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPSQHHHHHH  26 I1-GS10-E1: I/E tandem having I1VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV 8) and short tail) fused via GS₁₀YAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSGS linker (GS10 is SEQ ID NO: 11) toGSGSGS VSDVPRDLEVVAATPTSLLISWVAGAEDYQ E1 (with N-terminal extensionYYRITYGETGGNSPVQEFTVPHDLVTATISGLKPGV (N + 8) and no tail)DYTITVYAVTDMMHVEYTEHPISINYRT  27 I1-GS10-E1: I/E tandem having I1MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI (with N-terminal extenxion (N +TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI 10) and short tail) fused via GS₁₀TVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS linker (GS10 is SEQ ID NO: 11) toGSGSGSGS VSDVPRDLEVVAATPTSLLISWVAGAED E1 (with N-terminal extensionYQYYRITYGETGGNSPVQEFTVPHDLVTATISGLKP (N + 8) and Ser tail)GVDYTITVYAVTDMMHVEYTEHPISINYRTEIDKPS Q  28I1-GS10-E1: I/E tandem having I1 MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI(with N-terminal extension (N + TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI10) and short tail) fused via GS₁₀ TVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGSGS VSDVPRDLEVVAATPTSLLISWVAGAEDE1 (with N-terminal extension YQYYRITYGETGGNSPVQEFTVPHDLVTATISGLKP (N +8) and Ser tail with his tag) GVDYTITVYAVTDMMHVEYTEHPISINYRTEIDKPSQHHHHHH  29 E1-GS10-I1: E/I tandem having E1VSDVPRDLEVVAATPTSLLISWVAGAEDYQYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPHDLVTATISGLKPGVDYTITV 8) and short tail) fused via GS₁₀YAVTDMMHVEYTEHPISINYRTEIDK GSGSGSGSGS linker (GS10 is SEQ ID NO: 11) toGSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSARL I1 (with N-terminal extensionKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGL (N + 8) and no tail)KPGVDYTITVYAVTRFRDYQPISINYRT  30 E1-GS10-I1: E/I tandem having E1MGVSDVPRDLEVVAATPTSLLISWVAGAEDYQYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPHDLVTATISGLKPGVDYTI 10) and short tail) fused via GS₁₀TVYAVTDMMHVEYTEHPISINYRTEIDK GSGSGSGS linker (GS10 is SEQ ID NO: 11) toGSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSA I1 (with N-terminal extensionRLKVARYYRITYGETGGNSPVQEFTVPKNVYTATIS (N + 8) and Ser tail)GLKPGVDYTITVYAVTRFRDYQPISINYRTE IDKPS Q  31E1-GS10-I1: E/I tandem having E1 MGVSDVPRDLEVVAATPTSLLISWVAGAEDYQYYRI(with N-terminal extension (N + TYGETGGNSPVQEFTVPHDLVTATISGLKPGVDYTI10) and short tail) fused via GS₁₀ TVYAVTDMMHVEYTEHPISINYRTEIDK GSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSAI1 (with N-terminal extension RLKVARYYRITYGETGGNSPVQEFTVPKNVYTATIS (N +8) and Ser tail with his tag) GLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPSQHHHHHH  32 ¹⁰Fn3 scaffold, wherein the BC,VSDVPRDLEVVAATPTSLLI(X)_(n)YYRITYGETGG DE, and FG loops are representedNSPVQEFTV(X)_(o)ATISGLKPGVDYTITVYAV(X)_(p)by (X)_(n), (X)_(o), and (X)_(p), ISINYRTrespectively, and n is an integer from 1-20, o is an integer from1-20, and p is an integer from 1-40  33BC loop sequence from EGFR binder SWVAGAEDYQ E1  34BC loop sequence from EGFR binder X_(m)VAGAEDYQX_(n)E1, wherein X is any amino acid and m and n are independentlyselected from 0 to 5 amino acids  35 DE loop sequence from EGFR binderPHDLVT E1  36 DE loop sequence from EGFR binder X_(o)HDLVX_(p)E1, wherein X is any amino acid and o and p are independentlyselected from 0 to 5 amino acids  37 FG loop sequence from EGFR binderTDMMHVEYTEHP E1  38 FG loop sequence from EGFR binderX_(q)DMMHVEYTEHX_(r) E1, wherein X is any amino acidand q and r are independently selected from 0 to 5 amino acids  39BC loop sequence from EGFR binder SWDSGRGSYQ E2  40BC loop sequence from EGFR binder X_(g)DSGRGSYQX_(h)E2, wherein X is any amino acid and g and h are independentlyselected from 0 to 5 amino acids  41 DE loop sequence from EGFR binderPGPVHT E2  42 DE loop sequence from EGFR binder X_(i)GPVHX_(j)E2, wherein X is any amino acid and i and j are independentlyselected from 0 to 5 amino acids  43 FG loop sequence from EGFR binderTDHKPHADGPHTYHESP E2  44 FG loop sequence from EGFR binderX_(k)DHKPADGPHTYHEX_(l) E2, wherein X is any amino acidand k and l are independently selected from 0 to 5 amino acids  45BC loop sequence from IGF-IR SWSARLKVAR binder I1  46BC loop sequence from IGF-IR X_(a)SARLKVAX_(b)binder I1, wherein X is any amino acid and a and b are independ-ently selected from 0 to 5 amino acids  47 DE loop sequence from IGF-IRPKNVYT binder I1  48 DE loop sequence from IGF-IR X_(c)KNVYX_(d)binder I1, wherein X is any amino acid and c and d are independ-ently selected from 0 to 5 amino acids  49 FG loop sequence from IGF-IRTRFRDYQP binder I1  50 FG loop sequence from IGF-IR X_(e)RFRDYQX_(f)binder I1, wherein X is any amino acid and e and f are independ-ently selected from 0 to 5 amino acids  51 Linker GPG  52E3 EGFR monomer with N-terminal MGVSDVRPDLEVVAATPTSLLISWLPGKLRYQYYRIextension (N + 10), Ser tail and TYGETGGNSPVQEFTVPHDLRTATISGLKPGVDYTIhis tag TVYAVTNMMHVEYSEYPISINYRTEIDKPSQHHHHH H  53E3-GS10-I1: E/I tandem having E3 MGVSDVPRDLEVVAATPTSLLISWLPGKLRYQYYRI(with N-terminal extension (N + TYGETGGNSPVQEFTVPHDLRTATISGLKPGVDYTI10) and short tail) fused via GS₁₀ TVYAVTNMMHVEYSEYPISINYRTEIDK GSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSAI1 (with N-terminal extension RLKVARYYRITYGETGGNSPVQEFTVPKNVYTATIS (N +8) and Cys tail with his tag) GLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPCQHHHHHH  54 I1-GS10-E3: I/E tandem having I1MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI with N-terminal extension (N + 10)TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI and short tail) fused via GS₁₀TVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS linker (GS10 is SEQ ID NO: 11) toGSGSGSGS VSDVPRDLEVVAATPTSLLISWLPGKLR E3 (with N-terminal extensionYQYYRITYGETGGNSPVQEFTVPHDLRTATISGLKP (N + 8) and Cys tail with his tag)GVDYTITVYAVTNMMHVEYSEYPISINYRTEIDKPC QHHHHHH  55E1-GS10-I1: E/I tandem having E1 MGVSDVPRDLEVVAATPTSLLISWVAGAEDYQYYRI(with N-terminal extension (N + TYGETGGNSPVQEFTVPHDLVTATISGLKPGVDYTI10) and short tail) fused via GS₁₀ TVYAVTDMMHVEYTEHPISINYRTEIDK GSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSAI1 (with N-terminal extension RLKVARYYRITYGETGGNSPVQEFTVPKNVYTATIS (N +8) and Cys tail with his tag) GLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPCQHHHHHH  56 E2-GS10-I1: E/I tandem having E2MGVSDVPRDLEVVAATPTSLLISWDSGRGSYQYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPGPVHTATISGLKPGVDYTI 10) and short tail) fused via GS₁₀TVYAVTDHKPHADGPHTYHESPISINYRTEIDK GSG linker (GS10 is SEQ ID NO: 11) toSGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLL I1 (with N-terminal extensionISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVY (N + 8) and Cys tail with his tag)TATISGLKPGVDYTITVYAVTRFRDYQPISINYRTE IDKPCQHHHHHH  57I1-GS10-E1: I/E tandem having I1 MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI(with N-terminal extension (N + TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI10) and short tail) fused via GS₁₀ TVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGSGS VSDVPRDLEVVAATPTSLLISWVAGAEDE1 (with N-terminal extension YQYYRITYGETGGNSPVQEFTVPHDLVTATISGLKP (N +8) and Cys tail with his tag) GVDYTITVYAVTDMMHVEYTEHPISINYRTEIDKPCQHHHHHH  58 I1-GS10-E2: I/E tandem having I1MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI 10) and short tail) fused via GS₁₀TVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS linker (GS10 is SEQ ID NO: 11) toGSGSGSGS VSDVPRDLEVVAATPTSLLISWDSGRGS E2 (with N-terminal extensionYQYYRITYGETGGNSPVQEFTVPGPVHTATISGLKP (N + 8) and Cys tail with his tag)GVDYTITVYAVTDHKPHADGPHTYHESPISINYRTE IDKPCQHHHHHH  59BC loop sequence from EGFR binder SWLPGKLRYQ E3  60BC loop sequence from EGFR binder X_(s)LPGKLRYQX_(t)E3, wherein X is any amino acid and s and t are independentlyselected from 0 to 5 amino acids  61 DE loop sequence from EGFR binderPHDLRT E3  62 DE loop sequence from EGFR binder X_(u)HDLRX_(w)E3, wherein X is any amino acid and u and w are independentlyselected from 0 to 5 amino acids  63 FG loop sequence from EGFR binderTNMMHVEYSEYP E3  64 DE loop sequence from EGFR binderX_(y)NMMHVEYSEYX_(z) E3, wherein X is any amino acidand y and z are independently selected from 0 to 5 amino acids  65I1 IGF-IR monomer core sequence: EVVAATPTSLLISWSARLKVARYYRITYGETGGNSPI1 without N-terminal extension VQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDor C-terminal tail YQPISINYRT  66 E1 EGFR monomer core sequence: E1EVVAATPTSLLISWVAGAEDYQYYRITYGETGGNSP without N-terminal extension orVQEFTVPHDLVTATISGLKPGVDYTITVYAVTDMMH C-terminal tail VEYTEHPISINYRT  67E2 EGFR monomer core sequence: E2 EVVAATPTSLLISWDSGRGSYQYYRITYGETGGNSPwithout N-terminal extension or VQEFTVPGPVHTATISGLKPGVDYTITVYAVTDHKPC-terminal tail HADGPHTYHESPISINYRT  68 E3 EGFR monomer core sequence:EVVAATPTSLLISWLPGKLRYQYYRITYGETGGNSP SEQ ID NO: 82 without N-terminalVQEFTVPHDLRTATISLGKPGVDYTITVYAVTNMMH extension or C-terminal tailVEYSEYPISINYRT  69 Exemplary N-terminal extension MGVSDVPRDL (N + 10) 70 Exemplary N-terminal extension GVSDVPRDL  71Exemplary N-terminal extension VSDVPRDL (N + 8)  72Exemplary N-terminal extension, X_(n)SDVPRDLwherein X is any amino acid and n is 0, 1 or 2, preferably when n =1, X is Met or Gly and when n = 2, X is Met-Gly  73Exemplary N-terminal extension, X_(n)DVPRDLwherein X is any amino acid and n is 0, 1 or 2, preferably when n =1, X is Met or Gly and when n = 2, X is Met-Gly  74Exemplary N-terminal extension, X_(n)VPRDLwherein X is any amino acid and n is 0, 1 or 2, preferably when n =1, X is Met or Gly and when n = 2, X is Met-Gly  75Exemplary N-terminal extension, X_(n)PRDLwherein X is any amino acid and n is 0, 1 or 2, preferably when n =1, X is Met or Gly and when n = 2, X is Met-Gly  76Exemplary N-terminal extension, X_(n)RDL wherein X is any amino acid andn is 0, 1 or 2, preferably when n = 1, X is Met or Gly and when n =2, X is Met-Gly  77 Exemplary N-terminal extension, X_(n)DLwherein X is any amino acid and n is 0, 1 or 2, preferably when n =1, X is Met or Gly and when n = 2, X is Met-Gly  78 Short tail EIDK  79Exemplary C-terminal tail EIDKP  80 Exemplary C-terminal tail EIDKPS  81Exemplary C-terminal tail EIDKPC  82 E3 EGFR monomer with N-terminalVSDVPRDLEVVAATPTSLLISWLPGKLRYQYYRITY extension (N + 8) and no tailGETGGNSPVQEFTVPHDLRTATISGLKPGVDYTITV YAVTNMMHVEYSEYPISINYRT  87E3-GS10-I1: E/I tandem having E3 VSDVPRDLEVVAATPTSLLISWLPGKLRYQYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPHDLRTATISGLKPGVDYTITV8) and short tail) fused via GS₁₀ YAVTNMMHVEYSEYPISINYRTEIDK GSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSARLI1 (with N-terminal extension KVARYYRITYGETGGNSPVQEFTVPKNVYTATISGL (N +8) and Cys tail) KPGVDYTITVYAVTRFRDYQPISINYRTEIDKPCQ  88I1-GS10-E3: I/E tandem having I1 VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and short tail) fused via GS₁₀ YAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGS VSDVPRDLEVVAATPTSLLISWLPGKLRYQE3 (with N-terminal extension YYRITYGETGGNSPVQEFTVPHDLRTATISGLKPGV (N +8) and Cys tail) DYTITVYAVTNMMHVEYSEYPISINYRTEIDKPCQ  89I1-GS10-I1: E/I tandem having E1 VSDVPRDLEVVAATPTSLLISWVAGAEDYQYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPDHLVTATISLGKPGVDYTITV8) and short tail) fused via GS₁₀ YAVTDMMHVEYTEHPISINYRTEIDK GSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSARLI1 (with N-terminal extension KVARYYRITYGETGGNSPVQEFTVPKNVYTATISGL (N +8) and Cys tail) KPGVDYTITVYAVTRFRDYQPISINYRTEIDKPCQ  90E2-GS10-I1: E/I tandem having E2 VSDVPRDLEVVAATPTSLLISWDSGRGSYQYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPGPVHTATISGLKPGVDYTITV8) and short tail) fused via GS₁₀ YAVTDHKPHADGPHTYHESPISINYRTEIDK GSGSGlinker (GS10 is SEQ ID NO: 11) to SGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISI1 (with N-terminal extension WSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTA (N +8) and Cys tail) TISGLKPGVDYTITVYAVTRFRDYQPISINYRTEID KPCQ  91I1-GS10-E1: I/E tandem having I1 VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and short tail) fused via GS₁₀ YAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGS VSDVPRDLEVVAATPTSLLISWVAGAEDYQE1 (with N-terminal extension YYRITYGETGGNSPVQEFTVPHDLVTATISGLKPGV (N +8) and Cys tail) DYTITVYAVTDMMHVEYTEHPISINYRTEIDKPCQ  92I1-GS10-E2: I/E tandem having I1 VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and short tail) fused via GS₁₀ YAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGS VSDVPRDLEVVAATPTSLLISWDSGRGSYQE2 (with N-terminal extension YYRITYGETGGNSPVQEFTVPGPVHTATISGLKPGV (N +8) and Cys tail) DYTITVYAVTDHKPHADGPHTYHESPISINYRTEID KPCQ  93PA3 Linker PAPAPA  94 PA6 Linker PAPAPAPAPAPA  95 PA9 LinkerPAPAPAPAPAPAPAPAPA  96 Modified Ser tail EGSGS  97 Modified Cys tailEGSGC  98 E3-(PA)_(n)-I1: E/I tandem having E3VSDVPRDLEVVAATPTSLLISWLPGKLRYQYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPHDLRTATISGLKPGVDYTITV8) and an E tail) fused via (PA)_(n) YAVTNMMHVEYSEYPISINYRTE(PA)_(n)VSDVPRDL linker ((PA)_(n) is SEQ ID NO: 488)EVVAATPTSLLISWSARLKVARYYRITYGETGGNPS to I1 (with N-terminal extensionVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRD (N + 8) and a modified Ser or CysYQPISINYRTEGSGX tail), wherein n = 3, 6 or 9, and X = Ser or Cys  99I1-(PA)_(n)-E3: I/E tandem having I1VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and an E tail) fused via (PA)_(n) YAVTRFRDYQPISINYRTE(PA)_(n)VSDVPRDLEVVA linker ((PA)_(n) is SEQ ID NO: 488)ATPTSLLISWLPGKLRYQYYRITYGETGGNSPVQEF to E3 (with N-terminal extensionTVPHDLRTATISGLKPGVDYTITVYAVTNMMHVEYS (N + 8) and a modified Ser or CysEYPISINYRTEGSGX tail), wherein n = 3, 6 or 9, and X = Ser or Cys 100E1-(PA)_(n)-I1: E/I tandem having E1VSDVPRDLEVVAATPTSLLISWVAGAEDYQYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPDHLVTATISGLKPGVDYTITV8) and an E tail) fused via (PA)_(n) YAVTDMMHVEYTEHPISINYRTE(PA)_(n)VSDVPRDL linker ((PA)_(n) is SEQ ID NO: 488)EVVAATPTSLLISWSARLKVARYYRITYGETGGNSP to I1 (with N-terminal extensionVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRD (N + 8) and a modified Ser or CysYQPISINYRTEGSGX tail), wherein n = 3, 6 or 9, and X = Ser or Cys 101E2-(PA)_(n)-I1: E/I tandem having E2VSDVPRDLEVVAATPTSLLISWDSGRGSYQYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPGPVHTATISGLKPGVDYTITV8) and an E tail) fused via (PA)_(n)YAVTDHKPHADGPHTYHESPISINYRTE(PA)_(n) VSDlinker ((PA)_(n) is SEQ ID NO: 488) VPRDLEVVAATPTSLLISWSARLKVARYYRITYGETto I1 (with N-terminal extension GGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAV(N + 8) and a modified Ser or Cys TRFRDYQPISINYRTEGSGXtail), wherein n = 3, 6 or 9, and X = Ser or Cys 102I1-(PA)_(n)-E1: I/E tandem having I1VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and an E tail) fused via (PA)_(n) YAVTRFRDYQPISINYRTE(PA)_(n)VSDVPRDLEVVA linker ((PA)_(n) is SEQ ID NO: 488)ATPTSLLISWVAGAEDYQYYRITYGETGGNSPVQEF to E1 (with N-terminal extensionTVPHDLVTATISGLKPGVDYTITVYAVTDMMHVEYT (N + 8) and a modified Ser or CysEHPISINYRTEGSGX tail), wherein n = 3, 6 or 9, and X = Ser or Cys 103I1-(PA)_(n)-E2: I/E tandem having I1VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and an E tail) fused via (PA)_(n) YAVTRFRDYQPISINYRTE(PA)_(n)VSDVPRDLEVVA linker ((PA)_(n) is SEQ ID NO: 488)ATPTSLLISWDSGRGSYQYYRITYGETGGNSPVQEF to E2 (with N-terminal extensionTVPGPVHTATISGLKPGVDYTITVYAVTDHKPHADG (N + 8) and a modified Ser or CysPHTYHESPISINYRTEGSGX tail), wherein n = 3, 6 or 9, and X = Ser or Cys104 E3-GS10-E1: E/I tandem having E3VSDVPRDLEVVAATPTSLLISWLPGKLRYQYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPHDLRTATISGLKPGVDYTITV 8) and short tail) fused via GS₁₀YAVTNMMHVEYSEYPISINYRTEIDK GSGSGSGSGS linker (GS10 is SEQ ID NO: 11) toGSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSARL I1 (with N-terminal extensionKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGL (N + 8) and Ser tail)KPGVDYTITVYAVTRFRDYQPISINYRTEIDKPSQ 105 I1-GS10-E3: I/E tandem having I1VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPKNVYTATISLGKPGVDYTITV 8) and short tail) fused via GS₁₀YAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSGS linker (GS10 is SEQ ID NO: 11) toGSGSGS VSDVPRDLEVVAATPTSLLISWLPGKLRYQ E3 (with N-terminal extensionYYRITYGETGGNSPVQEFTVPHDLRTATISGLKPGV (N + 8) and Ser tail)DYTITVYAVTNMMHVEYSEYPISINYRTEIDKPSQ 106 E4 EGFR monomer with N-terminalVSDVPRDLEVVAATPTSLLISWHERDGSRQYYRITY extension (N + 8) and no tailGETGGNSPVQEFTVPGGVRTATISGLKPGVDYTITV YAVTDYFNPTTHEYIYQTTPISINYRT 107E4 EGFR monomer with N-terminal MGVSDVPRDLEVVAATPTSLLISWHERDGSRQYYRIextension (N + 10) and a Ser with TYGETGGNSPVQEFTVPGGVRTATISGLKPGVDYTIHis tag TVYAVTDYFNPTTHEYIYQTTPISINYRTEIDKPSQ HHHHHH 108E4 EGFR monomer core sequence: E4 EVVAATPTSLLISWHERDGSRQYYRITYGETGGNSPwithout N-terminal extension or VQEFTVPGGVRTATISGLKPGVDYTITVYAVTDYFNC-terminal tail PTTHEYIYQTTPISINYRT 109BC loop sequence from EGFR binder SWHERDGSRQ E4 110DE loop sequence from EGFR binder PGGVRT E4 111FG loop sequence from EGFR binder TDYFNPTTHEYIYQTTP E4 112E5 EGFR monomer with N-terminal VSDVPRDLEVVAATPTSLLISWWAPVDRYQYYRITYextension (N + 8) and no tail GETGGNSPVQEFTVPRDVYTATISGLKPGVDYTITVYAVTDYKPHADGPHTYHESPISINYRT 113 E5 EGFR monomer with N-terminalMGVSDVPRDLEVVAATPTSLLISWWAPVDRYQYYRI extension (N + 10) and a modifiedTYGETGGNSPVQEFTVPRDVYTATISGLKPGVDYTI Ser or Cys tail, wherein X = SerTVYAVTDYKPHADGPHTYHESPISINYRTEIDKPXQ or Cys; may optionally comprisea 6X His tag (SEQ ID NO: 487) 114 E5 EGFR monomer core sequence: E5EVVAATPTSLLISWWAPVDRYQYYRITYGETGGNSP without N-terminal extension orVQEFTVPRDVYTATISGLKPGVDYTITVYAVTDYKP C-terminal tail HADGPHTYHESPISINYRT115 BC loop sequence from EGFR binder SWWAPVDRYQ E5 116DE loop sequence from EGFR binder PRDVYT E5 117FG loop sequence from EGFR binder TDYKPHADGPHTYHESP E5 118E4-GS10-I1: E/I tandem having E4 VSDVPRDLEVVAATPTSLLISWHERDGSRQYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPGGVRTATISGLKPGVDYTITV8) and short tail) fused via GS10 YAVTDYFNPTTHEYIYQTTPISINYRTEIDK GSGSGlinker (GS10 is SEQ ID NO: 11) to SGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISI1 (with N-terminal extension WSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTA (N +8) and no tail) TISGLKPGVDYTITVYAVTRFRDYQPISINYRT 119E4-GS10-I1: E/I tandem having E4 VSDVPRDLEVVAATPTSLLISWHERDGSRQYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPGGVRTATISGLKPGVDYTITV8) and short tail) fused via GS10 YAVTDYFNPTTHEYIYQTTPISINYRTEIDK GSGSGlinker (GS10 is SEQ ID NO: 11) to SGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISI1 (with N-terminal extension WSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTA (N +8) and modified Ser or Cys TISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDtail), wherein X = Ser or Cys; KPXQ may optionally comprise a 6X Histag (SEQ ID NO: 487) 120 E4-GS10-I1: E/I tandem having E4MGVSDVPRDLEVVAATPTSLLISWHERDGSRQYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPGGVRTATISGLKRTEIDK G 10) and short tail) fused via GS10SGSGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTS linker (GS10 is SEQ ID NO: 11) toLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKN I1 (with N-terminal extensionVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYR (N + 8) and Cys tail) with his tagTEIDKPCQHHHHHH 121 E4-(PA)_(n)-I1: E/I tandem having E4VSDVPRDLEVVAATPTSLLISWHERDGSRQYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPGGVRTATISGLKPGVDYTITV8) and an E tail) fused via (PA)_(n)YAVTDYFNPTTHEYIYQTTPISINYRTE(PA)_(n) VSDlinker ((PA)_(n) is SEQ ID NO: 488) VPRDLEVVAATPTSLLISWSARLKVARYYRITYGETto I1 (with N-terminal extension GGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAV(N + 8) and modified Ser or Cys TRFRDYQPISINYRTEIDKPCQHHHHHHtail), wherein n = 3, 6 or 9, and X = Ser or Cys; may optionallycomprise a 6X His tag (SEQ ID NO: 487) 122I1-GS10-E4: I/E tandem having I1 VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and short tail) fused via GS10 YAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGS VSDVPRDLEVVAATPTSLLISWHERDGSRQE4 (having N-terminal extension YYRITYGETGGNSPVQEFTVPGGVRTATISGLKPGV(N + 8) and no tail) DYTITVYAVTDYFNPTTHEYIYQTTPISINYRT 123I1-GS10-E4: I/E tandem having I1 VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and short tail) fused via GS10 YAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGS VSDVPRDLEVVAATPTSLLISWHERDGSRQE4 (with N-terminal extension YYRITYGETGGNSPVQEFTVPGGVRTATISLGKPGV (N +8) and modified Ser or Cys DYTITVYAVTDYFNPTTHEYIYQTTPISINYRTEIDtail), wherein X = Ser or Cys; KPXQ may optionally comprise a 6X Histag (SEQ ID NO: 487) 124 I1-GS10-E4: I/E tandem having I1MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYR (with N-terminal extension (N +ITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDY 10) and short tail) fused via GS10TITVYAVTRFRDYQPISINYRTEIDK GSGSGSGSG linker (GS10 is SEQ ID NO: 11) toSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISWHE E4 (with N-terminal extensionRDGSRQYYRITYGETGGNSPVQEFTVPGGVRTATI (N + 8) and a Cys tail) with hisSGLKPGVDYTITVYAVTDYFNPTTHEYIYQTTPIS tag INYRTEIDKPCQHHHHHH 125I1-(PA)_(n)-E4: I/E tandem having I1 VSDVPRDLEVVAATPTSLLISWSARLKVARYYRIT(with N-terminal extension (N + YGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI8) and an E tail) fused via (PA)_(n) TVYAVTRFRDYQPISINYRTE(PA)_(n)VSDVPRDLE linker ((PA)_(n) is SEQ ID NO: 488)VVAATPTSLLISWHERDGSRQYYRITYGETGGNSP to E4 (with N-terminal extensionVQEFTVPGGVRTATISGLKPGVDYTITVYAVTDYF (N + 8) and modified Ser or CysNPTTHEYIYQTTPISINYRTEIDKPXQ tail), wherein n = 3, 6 or 9, and X =Ser or Cys; may optionally comprise a 6X His tag (SEQ ID NO: 487) 126E5-GS10-I1: E/I tandem having E5 VSDVPRDLEVVAATPTSLLISWWAPVDRYQYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPRDVYTATISGLKPGVDYTITV8) and short tail) fused via GS10 YAVTDYKPHADGPHTYHESPISINYRTEIDK GSGSGlinker (GS10 is SEQ ID NO: 11) to SGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISI1 (with N-terminal extension WSSARLKVARYYRITYGETGGNSPVQEFTNPKNVYT (N +8) and no tail) ATISGLKPGVDYTITVYAVTRFRDYQPISINYRT 127E5-GS10-I1: E/I tandem having E5 VSDVPRDLEVVAATPTSLLISWWAPVDRYQYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPRDVYTATISGLKPGVDYTITV8) and short tail) fused via GS10 YAVTDYKPHADGPHTYHESPISINYREIDK GSGSGlinker (GS10 is SEQ ID NO: 11) to SGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLLISI1 (with N-terminal extension WSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTA (N +8) and modified Ser or Cys TISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDtail), wherein X = Ser or Cys; KPXQ may optionally comprise a 6X Histag (SEQ ID NO: 487) 128 E5-GS10-I1: E/I tandem having E5MGVDSVPRDLEVVAATPTSLLISWWAPVDRYQYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPRDVYTATISGLKPGVDYTI 10) and short tail) fused via GS10TVYAVTDYKPHADGPHTYHESPISINYRTEIDK GSG linker (GS10 is SEQ ID NO: 11) toSGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLL I1 (with N-terminal extensionISWSARLKVARYYRITYGETGGNSPQEFTVPKNVYT (N + 8) and a Cys tail), with aATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEI His tag DKPCQHHHHHH 129E5-(PA)_(n)-I1: E/I tandem having E5VSDVPRDLEVVAATPTSLLISWWAPVDRYQYYRITY (with N-terminal extension (N +GETGGNSPVQEFTVPRDVYTATISGLKPGVDYTITV8) and an E tail) fused via (PA)_(n)YAVTDYKPHADGPHTYHESPISINYRTE(PA)_(n) VSDlinker ((PA)_(n) is SEQ ID NO: 488) VPRDLEVVAATPTSLLISWSARLKVARYYRITYGETto I1 (with N-terminal extension GGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAV(N + 8) and modified Ser or Cys TRFRDYQPISINYRTEIDKPXQtail), wherein n = 3, 6 or 9, and X = Ser or Cys; may be optionallycomprise a 6X His tag (SEQ ID NO: 487) 130I1-GS10-E5: I/E tandem having I1 VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and short tail) fused via GS10 YAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGSGSlinker (GS10 is SEQ ID NO: 11) to GSGSGS VSDVPRDLEVVAATPTSLLISWWAPVDRYQE5 (with N-terminal extension YYRITYGETGGNSPVQEFTVPRDVYTATISGLKPGV (N +8) and no tail) DYTITVYAVTDYKPHADGPHTYHESPISINYRT 131I1-GS10-E5: I/E tandem having I1 VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITY(with N-terminal extension (N + GETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITV8) and short tail) fused via GS10 YAVTRFRDYQPSISINYRTEIDK GSGSGSGSGSGSGlinker (GS10 is SEQ ID NO: 11) to SGSGSGS VSDVPRDLEVVAATPTSLLISWWAPVDRYE5 (with N-terminal extension QYYRITYGETGGNSPVQEFTVPRDVYTATISGLKPG (N +8) and modified Ser or Cys VDYTITVYAVTDYKPHADGPHTYHESPISINYRTEItail), wherein X = Ser or Cys; DKPXQ may optionally comprise a 6X Histag (SEQ ID NO: 487) 132 I1-GS10-E5: I/E tandem having I1MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI 10) and short tail) fused viaTVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS GS10 linker (GS10 is SEQ ID NO:GSGSGSGS VSDVPRDLEVVAATPTSLLISWWAPVDR 11) to E5 (with N-terminalYQYYRITYGETGGNSPVQEFTVPRDVYTATISGLKP extension (N + 8) and a Cys tail)GVDYTITVYAVTDYKPHADGPHTYHESPISINYRTE with a His tag IDKPCQHHHHHH 133I1-(PA)_(n)-E5: I/E tandem having I1MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI8) and an E tail) fused via (PA)_(n) TVYAVTRFRDYQPISINYRTE(PA)_(n)VSDVPRDLEV linker ((PA)_(n) is SEQ ID NO: 488)VAATPTSLLISWWAPVDRYQYYRITYGETGGNSPVQ to E5 (with N-terminal extensionEFTVPRDVYTATISGLKPGVDYTITVYAVTDYKPHA (N + 8) and modified Ser or CysDGPHTYHESPISINYRTEIDKPXQ tail), wherein n = 3, 6 or 9, and X =Ser or Cys; may optionally comprise a 6X His tag (SEQ ID NO: 487) 134BC loop sequence from EGFR binder X_(g)HERDGSRQX_(h)E4, wherein X is any amino acid and g and h are independentlyselected from 0 to 5 amino acids 135 DE loop sequence from EGFR binderX_(i)GGVRX_(j) E4, wherein X is any amino acidand i and j are independently selected from 0 to 5 amino acids 136FG loop sequence from EGFR binder X_(k)DYFNPTTHEYIYQTTX_(l)E4, wherein X is any amino acid and k and l are independentlyselected from 0 to 5 amino acids 137 BC loop sequence from EGFR binderX_(g)WAPVDRYQX_(h) E5, wherein X is any amino acidand g and h are independently selected from 0 to 5 amino acids 138DE loop sequence from EGFR binder X_(i)RDVYX_(j)E5, wherein X is any amino acid and i and j are independentlyselected from 0 to 5 amino acids 139 FG loop sequence from EGFR binderX_(k)DYKPHADGPHTYHESX_(l) E5, wherein X is any amino acidand k and l are independently selected from 0 to 5 amino acids 140E85 EGFR monomer with N-terminal MGVSDVPRDLEVVAATPTSLLISWTQGSTHYQYYRIextension (N + 10) and Ser tail TYGETGGNSPVQEFTVPGMVYTATISGLKPGVDYTIwith his tag TVYAVTDYRDRSTHEYKYRTTPISINYRTEIDKPSQ HHHHHH 141E85 EGFR monomer core: E85 EVVAATPTSLLISWTQGSTHYQYYRITYGETGGNSPmonomer without N-terminal VQEFTVPFGMVYTATISGLKPGVDYTITVYAVTDYFextension or C-terminal tail DRSTHEYKYRTTPISINYRT 142E85 EGFR monomer, wherein X₁ is X₁ EVVAATPTSLLISWTQGSTHYQYYRITYGETGGNselected from the group consist- SPVQEFTVPGMVYTATISLGKPGVDYTITVYAVTDYing of SEQ ID NOs: 69-77 and X₂ FDRSTHEYKYRTTPISINYRTX₂is selected from the group consisting of SEQ ID NOs: 9, 10,or 78-81; in exemplary embodi- ments, X₁ is SEQ ID NO: 69 or 71and X₂ is SEQ ID NO: 9 or 10; may optionally comprise a his tag 143BC loop sequence from EGFR binder SWTQGSTHYQ E85 144DE loop sequence from EGFR binder PGMVYT E85 145FG loop sequence from EGFR binder TDYFDRSTHEYKYRTTP E85 146BC loop sequence from EGFR binder X_(g)TQGSTHYQX_(h)E85, wherein X is any amino acid and g and h are independentlyselected from 0 to 5 amino acids 147 DE loop sequence from EGFR binderX_(i)GMVYX_(j) E85, wherein X is any amino acidand i and j are independently selected from 0 to 5 amino acids 148FG loop sequence from EGFR binder X_(k)DYFDRSTHEYKYRTTX_(l)E85, wherein X is any amino acid and k and l are independentlyselected from 0 to 5 amino acids 149 E85-GS10-I1: E/I tandem havingMGVSDVPRDLEVVAATPTSLLISWTQGSTHYQYYRI E85 (with N-terminal extensionTYGETGGNSPVQEFTVPGMVYTATISGLKPGVDYTI (N + 10) and a short tail) fusedTVYAVTDYFDRSTHEYKYRTTPISINYRTEIDK GSG via GS10 linker (GS10 is SEQ IDSGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLL NO: 11) to I1 (with N-terminalISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVY extension (N + 8) and Cys tail)TATISGLKPGVDYTITVYAVTRFRDYQPISINYRTE with a 6X His tag IDKPCQHHHHHH(SEQ ID NO: 487) 150 E85-GS10-I1 core, wherein X₁ is X₁EVVAATPTSLLISWTQGSTHYQYYRITYGETGGN optional and when present isSPVQEFTVPGMVYTATISGLKPGVDYTITVYAVTDY selected from the group consist-FDRSTHEYKYRTTPISINYRTEIDK GSGSGSGSGSG ing of SEQ ID NOs: 69-77, X₂ isSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSARLK optional and when present isVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLK selected from the group consist-PGVDYTITVYAVTRFRDYQPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or78-81, and n = 3, 6 or 9; in exemplary embodiments, X₁ isSEQ ID NO: 69 or 71 and X₂ is SEQ ID NO: 9 or 10 151E85-(PA)_(n)-I1 core, wherein X₁ is X₁EVVAATPTSLLISWTQGSTHYQYYRITYGETGGN optional and when present isSPVQEFTVPGMVYTATISGLKPGVDYTITVYAVTDY selected from the group consist-FDRSTHEYKYRTTPISINYRTE(PA)_(n) VSDVPRDLE ing of SEQ ID NOs: 69-77, X₂ isVVAATPTSLLISWSARLKVARYYRITYGETGGNSPV optional and when present isQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDY selected from the group consist-QPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ isSEQ ID NO: 9 or 10 152 I1-GS10-E85: I/E tandem having I1MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPKNVYTATISLGKPGVDYTI 10) and a short tail) fused viaTVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS GS10 linker (GS10 is SEQ ID NO:GSGSGSGS VSDVPRDLEVVAATPTSLLISWTQGSTH 11) to E85 (with N-terminalYQYYRITYGETGGNSPVQEFTVPGMVYTATISGLKP extension (N + 8) and Cys tail)GVDYTITVYAVTDYFDRSTHEYKYRTTPISINYRTE with a 6X His tag (SEQ ID NO:IDKPCQHHHHHH 487) 153 I1-GS10-E85 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTEIDK GSGSGSGSGSGSGSGSGSGS ing of SEQ ID NOs: 69-77, X₂ isVSDVPRDLEVVAATPTSLLISWTQGSTHYQYYRITY optional and when present isGETGGNSPVQEFTVPGMVYTATISGLKPGVDYTITV selected from the group consist-YAVTDYFDRSTHEYKYRTTPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or78-81, and n = 3, 6 or 9; in exemplary embodiments, X₁ isSEQ ID NO: 69 or 71 and X₂ is SEQ ID NO: 9 or 10 154I1-(PA)_(n)-E85 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTE(PA)_(n) VSDVPRDLEVVAATPTSL ing of SEQ ID NOs: 69-77, X₂ isLISWTQGSTHYQYYRITYGETGGNSPVQEFTVPGMV optional and when present isYTATISGLKPGVDYTITVYAVTDYFDRSTHEYKYRT selected from the group consist-TPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ isSEQ ID NO: 9 or 10 155 E90 EGFR monomer with N-terminalMGVSDVPRDLEVVAATPTSLLISWYWEGLPYQYYRI extension (N + 10) and Ser tailTYGETGGNSPVQEFTVPRDVNTATISGLKPGVDYTI with his tagTVYAVTDWYNPDTHEYIYHTIPISINYRTEIDKPSQ HHHHHH 156E90 EGFR monomer core: E90 EVVAATPTSLLISWYWEGLPYQYYRITYGETGGNSmonomer without N-terminal PVQEFTVPRDVNTATISGLKPGVDYTITVYAVTDWextension or C-terminal tail YNPDTHEYIYHTIPISINYRT 157E90 EGFR monomer, wherein X₁ is X₁ EVVAATPTSLLISWYWEGLPYQYYRITYGETGGselected from the group consist- NSPVQEFTVPRDVNTATISGLKPGVDYTITVYAVTing of SEQ ID NOs: 69-77 and X₂ DWYNPDTHEYIYHTIPISINYRTX₂is selected from the group consisting of SEQ ID NOs: 9, 10,or 78-81; in exemplary embodi- ments, X₁ is SEQ ID NO: 69 or 71and X₂ is SEQ ID NO: 9 or 10; may optionally comprise a his tag 158BC loop sequence from EGFR binder SWYWEGLPYQ E90 159DE loop sequence from EGFR binder PRDVNT E90 160FG loop sequence from EGFR binder TDWYNPDTHEYIYHTIP E90 161BC loop sequence from EGFR binder X_(g)YWEGLPYQX_(h)E90, wherein X is any amino acid and g and h are independentlyselected from 0 to 5 amino acids 162 DE loop sequence from EGFR binderX_(i)RDVNX_(j) E90, wherein X is any amino acidand i and j are independently selected from 0 to 5 amino acids 163FG loop sequence from EGFR binder X_(k)DWYNPDTHEYIYHTIX_(l)E90, wherein X is any amino acid and k and l are independentlyselected from 0 to 5 amino acids 164 E90-GS10-I1: E/I tandem havingMGVSDVPRDLEVVAATPTSLLISWYWEGLPYQYYRI E90 (with N-terminal extensionTYGETGGNSPVQEFTVPRDVNTATISGLKPGVDYTI (N + 10) and a short tail) fusedTVYAVTDWYNPDTHEYIYHTIPISINYRTEIDK GSG via GS10 linker (GS10 is SEQ IDSGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLL NO: 11) to I1 (with N-terminalISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVY extension (N + 8) and Cys tail)TATISGLKPGVDYTITVYAVTRFRDYQPISINYRTE with a 6X His tag (SEQ ID NO:IDKPCQHHHHHH 487) 165 E90-GS10-I1 core, wherein X₁ is X₁EVVAATPTSLLISWYWEGLPYQYYRITYGETGGN optional and when present isSPVQEFTVPRDVNTATISGLKPGVDYTITVYAVTDW selected from the group consist-YNPDTHEYIYHTIPISINYRTEIDK GSGSGSGSGSG ing of SEQ ID NOs: 69-77, X₂ isSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSARLK optional and when present isVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLK selected from the group consist-PGVDYTITVYAVTRFRDYQPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or78-81, and n = 3, 6 or 9; in exemplary embodiments, X₁ is SEQID NO: 69 or 71 and X₂ is SEQ ID NO: 9 or 10 166E90-(PA)_(n)-I1 core, wherein X1 is X₁EVVAATPTSLLISWYWEGLPYQYYRITYGETGGN optional and when present isSPVQEFTVPRDVNTATISGLKPGVDYTITVYAVTDW selected from the group consist-YNPDTHEYIYHTIPISINYRTE(PA)_(n) VSDVPRDLE ing of SEQ ID NOs: 69-77, X₂ isVVAATPTSLLISWSARLKVARYYRITYGETGGNSPV optional and when present isQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDY selected from the group consist-QPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ is SEQ IDNO: 9 or 10 167 I1-GS10-E90: I/E tandem having I1MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI 10) and a short tail) fused viaTVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS GS10 linker (GS10 is SEQ ID NO:GSGSGSGS VSDVPRDLEVVAATPTSLLISWYWEGLP 11) to E90 (with N-terminalYQYYRITYGETGGNSPVQEFTVPRDVNTATISGLKP extension (N + 8) and Cys tail)GVDYTITVYAVTDWYNPDTHEYIYHTIPISINYRTE with a 6X His tag (SEQ ID NO:IDKPCQHHHHHH 487) 168 I1-GS10-E90 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTEIDK GSGSGSGSGSGSGSGSGSGS ing of SEQ ID NOs: 69-77, X₂ isVSDVPRDLEVVAATPTSLLISWYWEGLPYQYYRITY optional and when present isGETGGNSPVQEFTVPRDVNTATISGLKPGVDYTITV selected from the group consist-YAVTDWYNPDTHEYIYHTIPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or78-81, and n = 3, 6 or 9; in exemplary embodiments, X₁ is SEQID NO: 69 or 71 and X₂ is SEQ ID NO: 9 or 10 169I1-(PA)_(n)-E90 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTE(PA)_(n) VSDVPRDLEVVAATPTSL ing of SEQ ID NOs: 69-77, X₂ isLISWYWEGLPYQYYRITYGETGGNSPVQEFTVPRDV optional and when present isNTATISGLKPGVDYTITVYAVTDWYNPDTHEYIYHT selected from the group consist-IPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ is SEQ IDNO: 9 or 10 170 E96 EGFR monomer with N-terminalMGVSDVPRDLEVVAATPTSLLISWASNRGTYQYYRI extension (N + 10) and Ser tailTYGETGGNSPVQEFTVPGGVSTATISGLKPGVDYTI with his tagTVYAVTDAFNPTTHEYNYFTTPISINYRTEIDKPSQ HHHHHH 171E96 EGFR monomer core: E96 EVVAATPTSLLISWASNRGTYQYYRITYGETGGNSPmonomer without N-terminal VQEFTVPGGVSTATISGLKPGVDYTITVYAVTDAFNextension or C-terminal tail PTTHEYNYFTTPISINYRT 172E96 EGFR monomer, wherein X₁ is X₁ EVVAATPTSLLISWASNRGTYQYYRITYGETGGNselected from the group consist- SPVQEFTVPGGVVSTATISGLKPGVDYTITVYAVTDing of SEQ ID NOs: 69-77 and X₂ AFNPTTHEYNYFTTPISINYRTX₂is selected from the group consisting of SEQ ID NOs: 9, 10,or 78-81; in exemplary emobid- ments, X₁ is SEQ ID NO: 69 or 71and X₂ is SEQ ID NO: 9 or 10; may optionally comprise a his tag 173BC loop sequence from EGFR binder SWASNRGTYQ E96 174DE loop sequence from EGFR binder PGGVST E96 175FG loop sequence from EGFR binder TDAFNPTTHEYNYFTTP E96 176BC loop sequence from EGFR binder X_(g)ASNRGTYQX_(h)E96, wherein X is any amino acid and g and h are independentlyselected from 0 to 5 amino acids 177 DE loop sequence from EGFR binderX_(i)GGVSX_(j) E96, wherein X is any amino acidand i and j are independently selected from 0 to 5 amino acids 178FG loop sequence from EGFR binder X_(k)DAFNPTTHEYNYFTTX_(l)E96, wherein X is any amino acid and k and l are independentlyselected from 0 to 5 amino acids 179 E96-GS10-I1: E/I tandem havingMGVSDVPRDLEVVAATPTSLLISWASNRGTYQYYRI E96 (with N-terminal extensionTYGETGGNSPVQEFTVPGGVSTATISGLKPGVDYTI (N + 10) and a short tail) fusedTVYAVTDAFNPTTHEYNYFTTPISINYRTEIDK GSG via GS10 linker (GS10 is SEQ IDSGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLL NO: 11) to I1 (with N-terminalISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVY extension (N + 8) and Cys tail)TATISGLKPGVDYTITVYAVTRFRDYQPISINYRTE with a 6X His tag (SEQ ID NO:IDKPCQHHHHHH 487) 180 E96-GS10-I1 core, wherein X₁ is X₁EVVAATPTSLLISWASNRGTYQYYRITYGETGGN optional and when present isSPVQEFTVPGGVSTATISGLKPGVDYTITVYAVTDA selected from the group consist-FNPTTHEYNYFTTPISINYRTEIDK GSGSGSGSGSG ing of SEQ ID NOs: 69-77, X₂ isSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSARLK optional and when present isVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLK selected from the group consist-PGVDYTITVYAVTRFRDYQPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or78-81, and n = 3, 6 or 9; in exemplary embodiments, X₁ is SEQID NO: 69 or 71 and X₂ is SEQ ID NO: 9 or 10 181E96-(PA)_(n)-I1 core, wherein X1 is X₁EVVAATPTSLLISWASNRGTYQYYRITYGETGGN optional and when present isSPVQEFTVPGGVSTATISGLKPGVDYTITVYAVTDA selected from the group consist-FNPTTHEYNYFTTPISINYRTE(PA)_(n) VSDVPRDLE ing of SEQ ID NOs: 69-77, X₂ isVVAATPTSLLISWSARLKVARYYRITYGETGGNSPV optional and when present isQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDY selected from the group consist-QPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ is SEQ IDNO: 9 or 10 182 I1-GS10-E96: I/E tandem having I1MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI (with N-terminal extension (N +TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI 10) and a short tail) fused viaTVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS GS10 linker (GS10 is SEQ ID NO:GSGSGSGS VSDVPRDLEVVAATPTSLLISWASNRGT 11) to E96 (with N-terminalYQYYRITYGETGGNSPVQEFTVPGGVSTATISGLKP extension (N + 8) and Cys tail)GVDYTITVYAVTDAFNPTTHEYNYFTTPISINYRTE with a 6X His tag (SEQ ID NO:IDKPCQHHHHHH 487) 183 I1-GS10-E96 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTEIDK GSGSGSGSGSGSGSGSGSGS ing of SEQ ID NOs: 69-77, X₂ isVSDVPRDLEVVAATPTSLLISWASNRGTYQYYRITY optional and when present isGETGGNSPVQEFTVPGGVSTATISGLKPGVDYTITV selected from the group consist-YAVDAFNPTTHEYNYFTTPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n =3, 6 or 9; in exemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ isSEQ ID NO: 9 or 10 184 I1-(PA)_(n)-E96 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTE(PA)_(n) VSDVPRDLEVVAATPTSL ing of SEQ ID NOs: 69-77, X₂ isLISWASNRGTYQYYRITYGETGGNSPVQEFTVPGGV optional and when present isSTATISGLKPGVDYTITVYAVTDAFNPTTHEYNYFT selected from the group consist-TPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ isSEQ ID NO: 9 or 10 185 E105 EGFR monomer with N-terminalMGVSDVPRDLEVVAATPTSLLISWDAPTSRYQYYRI extension (N + 10) and Ser tailTYGETGGNSPVQEFTVPGGLSTATISLGKPGVDYTI with his tagTVYAVTDYKPHADGPHTYHESPISINYRTEIDKPSQ HHHHHH 186E105 EGFR monomer core: E105 EVVAATPTSLLISWDAPTSRYQYYRITYGETGGNSPmonomer without N-terminal VQEFTVPGGLSTATISGLKPGVDYTITVYAVTDYKPextension or C-terminal tail HADGPHTYHESPISINYRT 187E105 EGFR monomer, wherein X₁ is X₁ EVVAATPTSLLISWDAPTSRYQYYRITYGETGGNselected from the group consist- SPVQEFTVPGGLSTATISGLKPGVDYTITVYAVTDYing of SEQ ID NOs: 69-77 and X₂ KPHADGPHTYHESPISINYRTX₂is selected from the group consisting of SEQ ID NOs: 9, 10,or 78-81; in exemplary embodi- ments, X₁ is SEQ ID NO: 69 or 71and X₂ is SEQ ID NO: 9 or 10; may optionally comprise a his tag 188BC loop sequence from EGFR binder SWDAPTSRYQ E105 189DE loop sequence from EGFR binder PGGLST E105 117FG loop sequence from EGFR binder TDYKPHADGPHTYHESP E105 190BC loop sequence from EGFR binder X_(g)DAPTSRYQX_(h)E105, wherein X is any amino acid and g and h are independentlyselected from 0 to 5 amino acids 191 DE loop sequence from EGFR binderX_(i)GGLSX_(j) E105, wherein X is any amino acidand i and j are independently selected from 0 to 5 amino acids 139FG loop sequence from EGFR binder X_(k)DYKPHADGPHTYHESX_(l)E105, wherein X is any amino acid and k and l are independentlyselected from 0 to 5 amino acids 192 E105-GS10-I1: E/I tandem havingMGVSDVPRDLEVVAATPTSLLISWDAPTSRYQYYRI E105 (with N-terminal extensionTYGETGGNSPVQEFTVPGGLSTATISGLKPGVDYTI (N + 10) and a short tail) fusedTVYAVTDYKPHADGPHTYHESPISINYRTEIDK GSG via GS10 linker (GS10 is SEQ IDSGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLL NO: 11) to I1 (with N-terminalISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVY extension (N + 8) and Cys tail)TATISGLKPGVDYTITVYAVTRFRDYQPISINYRTE with a 6X His tag (SEQ ID NO:IDKPCQHHHHHH 487) 193 E105-GS10-I1 core, wherein X₁ is X₁EVVAATPTSLLISWDAPTSRYQYYRITYGETGGN optional and when present isSPVQEFTVPGGLSTATISGLKPGVDYTITVYAVTDY selected from the group consist-KPHADGPHTYHESPISINYRTEIDK GSGSGSGSGSG ing of SEQ ID NOs: 69-77, X₂ isSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSARLK optional and when present isVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLK selected from the group consist-PGVDYTITVYAVTRFRDYQPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or78-81, and n = 3, 6 or 9; in exemplary embodiments, X₁ isSEQ ID NO: 69 or 71 and X₂ is SEQ ID NO: 9 or 10 194E105-(PA)_(n)-I1 core, wherein X1 is X₁EVVAATPTSLLISWDAPTSRYQYYRITYGETGGN optional and when present isSPVQEFTVPGGLSTATISLGKPGVDYTITVYAVTDY selected from the group consist-KPHADGPHTYHESPISINYRTE(PA)_(n) VSDVPRDLE ing of SEQ ID NOs: 69-77, X₂ isVVAATPTSLLISWSARLKVARYYRITYGETGGNSPV optional and when present isQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDY selected from the group consist-QPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ isSEQ ID NO: 9 or 10 195 I1-GS10-E105: I/E tandem havingMGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI I1 (with N-terminal extensionTYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI (N + 10) and a short tail) fusedTVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS via GS10 linker (GS10 is SEQ IDGSGSGSGS VSDVPRDLEVVAATPTSLLISWDAPTSR NO: 11) to E105 (with N-terminalYQYYRITYGETGGNSPVQEFTVPGGLSTATISGLKP extension (N + 8) and Cys tail)GVDYTITVYAVTDYKPHADGPHTYHESPISINYRTE with a 6X His tag (SEQ ID NO:IDKPCQHHHHHH 487) 196 I1-GS10-E105 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTEIDK GSGSGSGSGSGSGSGSGSGS ing of SEQ ID NOs: 69-77, X₂ isVSDVPRDLEVVAATPTSLLISWDAPTSRYQYYRITY optional and when present isGETGGNSPVQEFTVPGGLSTATISGLKPGVDYTITV selected from the group consist-YAVTDYKPHADGPHTYHESPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or78-81, and n = 3, 6 or 9; in exemplary embodiments, X₁ isSEQ ID NO: 69 or 71 and X₂ is SEQ ID NO: 9 or 10 197I1-(PA)_(n)-E105 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTE(PA)_(n) VSDVPRDLEVVAATPTSL ing of SEQ ID NOs: 69-77, X₂ isLISWDAPTSRYQYYRITYGETGGNSPVQEFTVPGGL optional and when present isSTATISGLKPGVDYTITVYAVTDYKPHADGPHTYHE selected from the group consist-SPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ isSEQ ID NO: 9 or 10 198 E112 EGFR monomer with N-terminalMGVSDVPRDLEVVAATPTSLLISWDAGAVTYQYYRI extension (N + 10) and Ser tailTYGETGGNSPVQEFTVPGGVRTATISGLKPGVDYTI with his tagTVYAVTDYKPHADGPHTYHEYPISINYRTEIDKPSQ HHHHHH 199E112 EGFR monomer core: E112 EVVAATPTSLLISWDAGAVTYQYYRITYGETGGNSPmonomer without N-terminal VQEFTVPGGVRTATISGLKPGVDYTITVYAVTDYKPextension or C-terminal tail HADGPHTYHEYPISINYRT 200E112 EGFR monomer, wherein X₁ is X₁ EVVAATPTSLLISWDAGAVTYQYYRITYGETGGNselected from the group consist- SPVQEFTVPGGVRTATISGLKPGVDYTITVYAVTDYing of SEQ ID NOs: 69-77 and X₂ KPHADGPHTYHEYPISINYRTX₂is selected from the group consisting of SEQ ID NOs: 9, 10,or 78-81; in exemplary embodi- ments, X₁ is SEQ ID NO: 69 or 71and X₂ is SEQ ID NO: 9 or 10; may optionally comprise a his tag 201BC loop sequence from EGFR binder SWDAGAVTYQ E112 110DE loop sequence from EGFR binder PGGVRT E112 202FG loop sequence from EGFR binder TDYKPHADGPHTYHEYP E112 203BC loop sequence from EGFR binder X_(g)DAGAVTYQX_(h)E112, wherein X is any amino acid and g and h are independentlyselected from 0 to 5 amino acids 135 DE loop sequence from EGFR binderX_(i)GGVRX_(j) E112, wherein X is any amino acidand i and j are independently selected from 0 to 5 amino acids 204FG loop sequence from EGFR binder X_(k)DYKPHADGPHTYHEYX_(l)E112, wherein X is any amino acid and k and l are independentlyselected from 0 to 5 amino acids 205 E112-GS10-I1: E/I tandem havingMGVSDVPRDLEVVAATPTSLLISWDAGAVTYQYYRI E112 (with N-terminal extensionTYGETGGNSPVQEFTVPGGVRTATISGLKPGVDYTI (N + 10) and a short tail) fusedTVYAVTDYKPHADGPHTYHEYPISINYRTEIDK GSG via GS10 linker (GS10 is SEQ IDSGSGSGSGSGSGSGSGS VSDVPRDLEVVAATPTSLL NO: 11) to I1 (with N-terminalISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVY extension (N + 8) and Cys tail)TATISGLKPGVDYTITVYAVTRFRDYQPISINYRTE with a 6X His tag (SEQ ID NO:IDKPCQHHHHHH 487) 206 E112-GS10-I1 core, wherein X₁ is X₁EVVAATPTSLLISWDAGAVTYQYYRITYGETGGN optional and when present isSPVQEFTVPGGVRTATISGLKPGVDYTITVYAVTDY selected from the group consist-KPHADGPHTYHEYPISINYRTEIDK GSGSGSGSGSG ing of SEQ ID NOs: 69-77, X₂ isSGSGSGSGS VSDVPRDLEVVAATPTSLLISWSARLK optional and when present isVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLK selected from the group consist-PGVDYTITVYAVTRFRDYQPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or78-81, and n = 3, 6 or 9; in exemplary embodiments, X₁ isSEQ ID NO: 69 or 71 and X₂ is SEQ ID NO: 9 or 10 207E112-(PA)_(n)-I1 core, wherein X1 is X₁EVVAATPTSLLISWDAGAVTYQYYRITYGETGGN optional and when present isSPVQEFTVPGGVRTATISGLKPGVDYTITVYAVTDY selected from the group consist-KPHADGPHTYHEYPISINYRTE(PA)_(n) VSDVPRDLE ing of SEQ ID NOs: 69-77, X₂ isVVAATPTSLLISWSARLKVARYYRITYGETGGNSPV optional and when present isQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDY selected from the group consist-QPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ isSEQ ID NO: 9 or 10 208 I1-GS10-E112: I/E tandem havingMGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI I1 (with N-terminal extensionTYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI (N + 10) and a short tail) fusedTVYAVTRFRDYQPISINYRTEIDK GSGSGSGSGSGS via GS10 linker (GS10 is SEQ IDGSGSGSGS VSDVPRDLEVVAATPTSLLISWDAGAVT NO: 11) to E112 (with N-terminalYQYYRITYGETGGNSPVQEFTVPGGVRTATISGLKP extension (N + 8) and Cys tail)GVDYTITVYAVTDYKPHADGPHTYHEYPISINYRTE with a 6X His tag (SEQ ID NO:IDKPCQHHHHHH 487) 209 I1-GS10-E112 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTEIDK GSGSGSGSGSGSGSGSGSGS ing of SEQ ID NOs: 69-77, X₂ isVSDVPRDLEVVAATPTSLLISWDAGAVTYQYYRITY optional and when present isGETGGNSPVQEFTVPGGVRTATISGLKPGVDYTITV selected from the group consist-YAVTDYKPHADGPHTYHEYPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or78-81, and n = 3, 6 or 9; in exemplary embodiments, X₁ isSEQ ID NO: 69 or 71 and X₂ is SEQ ID NO: 9 or 10 210I1-(PA)_(n)-E112 core, wherein X₁ is X₁EVVAATPTSLLISWSARLKVARYYRITYGETGGN optional and when present isSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRF selected from the group consist-RDYQPISINYRTE(PA)_(n) VSDVPRDLEVVAATPTSL ing of SEQ ID NOs: 69-77, X₂ isLISWDAGAVTYQYYRITYGETGGNSPVQEFTVPGGV optional and when present isRTATISGLKPGVDYTITVYAVTDYKPHADGPHTYHE selected from the group consist-YPISINYRTX₂ ing of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; inexemplary embodiments, X₁ is SEQ ID NO: 69 or 71 and X₂ isSEQ ID NO: 9 or 10 211 I1-GSGCGS8-E5: I/E tandem havingMGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI I1 (with N-terminal extensionTYGETGGNSPVQEFTVNPKVYTATISGLKPGVDYTI (N + 10) and a modified shortTVYAVTRFRDYQPISINYRTEIEK GSGCGSGSGSGS tail) fused via GSGCGS8 linkerGSGSGSGS VSDVPRDLEVVAATPTSLLISWWAPVDR (GSGCGS8 is SEQ ID NO: 218) toYQYYRITYGETGGNSPVQEFTVPRDVYTATISGLKP E5 (with N-terminal extensionGVDYTITVYAVTDYKPHADGPHTYHESPISINYRTE (N + 8) and an E tail) with anHHHHHH optional 6X His tag (SEQ ID NO: 487) 212I1-GS10-E5-GSGC: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRIhaving I1 (with N-terminal TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTIextension (N + 10) and a modified TVYAVTRFRDYQPISINYRTEIEK GSGSGSGSGSGSshort tail) fused via GS10 linker GSGSGSGS VSDVPRDLEVVAATPTSLLISWWAPVDR(GS10 is SEQ ID NO: 11) to E5 YQYYRITYGETGGNSPVQEFTVPRDVYTATISGLKP(with N-terminal extension (N + GVDYTITVYAVTDYKPHADGPHTYHESPISINYRTE8) and a modified Cys tail) with GSGCHHHHHHan optional 6X His tag (SEQ ID NO: 487) 213 I1(S62C)-GS10-E5: I/E tandemMGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI having I1 (with N-terminalTYGETGGNSPVQEFTVPKNVYTATI

GLKPGVDY extension (N + 10), and S62C TITVYAVTRFRDYQPISINYRTEIEKGSGSGSGSGS substitution (boxed), and aGSGSGSGSGSVSDVPRDLEVVAATPTSLLISWWAPV modified short tail) fused viaDRYQYYRITYGETGGNSPVQEFTVPRDVYTATISGL GS10 linker (GS10 is SEQ ID NO:KPGVDYTITVYAVTDYKPHADGPHTYHESPISINYR 11) to E5 (with N-terminal TEHHHHHHextension (N + 8) and an E tail) with an optional 6X His tag (SEQID NO: 487); position 62 refers to the amino acid correspondingto position 62 of SEQ ID NO: 1 214 I1-GS10-E5(S62C): I/E tandemMGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI having I1 (with N-terminalTYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI extension (N + 10) and a modifiedTVYAVTRFRDYQPISINYRTEIEK GSGSGSGSGSGS short tail) fused via GS10 linkerGSGSGSGS VSDVPRDLEVVAATPTSLLISWWAPVDR (GS10 is SEQ ID NO: 11) to E5YQYYRITYGETGGNSPVQEFTVPRDVYTATI

GLK (with N-terminal extension (N + PGVDYTITVYAVTDYKPHADGPHTYHESPISINYRT8), and S62C substitution  EHHHHHH (boxed), and an E tail) with anoptional 6X His tag (SEQ ID NO: 487); position 62 refers to theamino acid corresponding to position 62 of SEQ ID NO: 1 215I1(S91C)-GS10-E5: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRIhaving I1 (with N-terminal TYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTIextension (N + 10), an S91C TVYAVTRFRDYQPI

NYRTEIEK GSGSGSGSGSGS substitution (boxed), and a GSGSGSGSVSDVPRDLEVVAATPTSLLISWWAPVDR modified short tail) fused viaYQYYRITYGETGGNSPVQEFTVPRDVYTATISGLKP GS10 linker (GS10 is SEQ ID NO:GCDYTITVYAVTDYKPHADGPHTYHESPISINYRTE 11) to E5 (with N-terminal HHHHHHextension (N + 8) and an E tail) with an optional 6X His tag (SEQID NO: 487); position 91 refers to the amino acid correspondingto position 91 of SEQ ID NO: 1 216 I1-GS10-E5(S91C): I/E tandemMGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRI having I1 (with N-terminalTYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTI extension (N + 10) and a modifiedTVYAVTRFRDYQPISINYRTEIEK GSGSGSGSGSGS short tail) fused via GS10GSGSGSGS VSDVPRDLEVVAATPTSLLISWWAPVDR linker (GS10 is SEQ ID NO: 11) toYQYYRITYGETGGNSPVQEFTVPRDVYTATISGLKP E5 (with N-terminal extensionGVDYTITVYAVTDYKPHADGPHTYHESPI

INYRT (N + 8), and S91C substitution EHHHHHH(boxed), and an E tail) with an optional 6X His tag (SEQ ID NO:487); position 91 refers to the amino acid corresponding toposition 91 of SEQ ID NO: 1 217 Modified short tail EIEK 218GSGCGS8 Linker GSGCGSGSGSGSGSGSGSGS

Example 1 In Cell Western Assay to Screen for EGFR Activity

In Cell Western assays were developed to screen various single ¹⁰Fn3clones for the ability to inhibit EGFR activity in order to identifythose that could be linked with IGF1R¹⁰Fn3 binders to construct E/Ibinders. In Cell Western assays were also used to screen and determinerelative potency of specific E/I ¹⁰Fn3 binders. Two In Cell Westernassays were developed to measure 1) inhibition of EGF-stimulated EGFRphosphorylation or 2) inhibition of EGF-stimulated ERK phosphorylation.Cells were seeded into poly-D-lysine coated 96-well microtiter plates(Becton Dickinson, Franklin Lakes, N.J.) at 24,000 cells/well for A431epidermoid carcinoma or FaDu head & neck carcinoma cells and allowed toadhere overnight. Cells were washed once and then incubated for 24 hoursin serum free media. Serial dilutions of the ¹⁰Fn3-based binders werenext applied to the cells and incubated for 2-3 hours prior tostimulation with 100 ng/ml EGF for 10 minutes. Following stimulation,cells were fixed for 20 minutes in PBS containing 3.7% formaldehyde andthen permeabilized in PBS containing 0.1% triton-X-100 for 15 minutes.Cells were blocked for one hour in Odyssey blocker (Li-Cor Biosciences,Lincoln, Nebr.) and incubated with antibodies to detect either EGFRphosphorylated on tyrosine 1068 (Cell Signaling, Beverly, Mass.) andβ-actin (Sigma, St. Louis, Mo.) or pERK (MAP kinase phosphorylated ontyrosine 202/threonine 204) and total ERK (Santa Cruz Biotechnology,Santa Cruz, Calif.). After washing three times in PBS containing 0.1%tween-20, secondary antibodies were added (Invitrogen, Carlsbad, Calif.or Rockland, Gilbertsville, Pa.). Cells were washed three times in PBScontaining 0.1% tween-20 and imaged on a Li-Cor Odyssey Infrared ImagingSystem (Li-Cor Biosciences, Lincoln, Nebr.). Each clone was assayed induplicate or triplicate and values were normalized to β-actin for thepEGFR assay and total ERK for the pERK assay. IC50 values werecalculated from linear regression analysis of percent inhibition ofmaximum signal minus background.

Results yielded various ¹⁰Fn3 clones that had ability to inhibitactivity of EGFR, and showed that certain specific E/I ¹⁰Fn3 binderspossessed similar activity to the example shown in FIG. 9.

Example 2 Expression of ¹⁰Fn3-Based Binders

E/I binders were produced by covalently linking an EGFR-binding ¹⁰Fn3 toan IGFIR-binding ¹⁰Fn3 using a glycine-serine linker, thereby generating¹⁰Fn3 dimers, wherein each ¹⁰Fn3 domain binds to a different target. TheIGFIR-binding ¹⁰Fn3 (I1) was previously described as SEQ ID NO: 226 inPCT Publication No. WO 2008/066752. Two novel EGFR-binding ¹⁰Fn3 (E2 andE1) were identified by screening an RNA-protein fusion library, asdescribed in PCT Publication No. WO 2008/066752, for binders to EGFR-Fc(R&D Systems, Minneapolis, Minn.). The following examples describeresults using a variety of His-tagged E/I ¹⁰Fn3-based binders(non-pegylated): E2-GS10-I1 (SEQ ID NO: 25), E1-GS10-I1 (SEQ ID NO: 31),I1-GS10-E1 (SEQ ID NO: 28), and I1-GS10-E2 (SEQ ID NO: 22).

The following examples also describe results with the followingpegylated, His-tagged E/I ¹⁰Fn3-based binders: E1-GS10-I1 (SEQ ID NO:55), E2-GS10-I1 (SEQ ID NO: 56), E3-GS10-I1 (SEQ ID NO: 53), I1-GS10-E1(SEQ ID NO: 57), I1-GS10-E2 (SEQ ID NO: 58), I1-GS10-E3 (SEQ ID NO: 54),E4-GS10-I1 (SEQ ID NO: 120), I1-GS10-E4 (SEQ ID NO: 124), E5-GS10-I1(SEQ ID NO: 128), I1-GS10-E5 (SEQ ID NO: 132), E85-GS10-I1 (SEQ ID NO:149), I1-GS10-E85 (SEQ ID NO: 152), E90-GS10-I1 (SEQ ID NO: 164),E96-GS10-I1 (SEQ ID NO: 179), E105-G51041 (SEQ ID NO: 192), I1-GS10-E105(SEQ ID NO: 195), E112-G51041 (SEQ ID NO: 205), I1-GS10-E112 (SEQ ID NO:208), I1-GSGCGS8-E5 (SEQ ID NO: 211), I1-GS10-E5-GSGC (SEQ ID NO: 212),I1(S62C)-GS10-E5 (SEQ ID NO: 213), I1-GS10-E5(S62C) (SEQ ID NO: 214),I1(S91C)-GS10-E5 (SEQ ID NO: 215), and I1-GS10-E5(S91C) (SEQ ID NO:216).

The examples also describe results using a His-tagged IGFRIR¹⁰Fn3-basedbinder, I1 (SEQ ID NO: 4), and ten His-tagged EGFR ¹⁰Fn3-based binders,E2 (SEQ ID NO: 6), E1 (SEQ ID NO: 8), E3 (SEQ ID NO: 52), E4 (SEQ ID NO:107), ES (SEQ ID NO: 113, wherein X=Ser and with a His tag at theC-terminus), E5 pegylated (SEQ ID NO: 113, wherein X=Cys and with a Histag at the C-terminus), E85 (SEQ ID NO: 140), E90 (SEQ ID NO: 155), E96(SEQ ID NO: 170), E105 (SEQ ID NO: 185), and E112 (SEQ ID NO: 198).Examples 32 also describes a variety of E monomers having the sequencesset forth in FIG. 45 and including a His tag at the C-terminus.

The various ¹⁰Fn3-based binders were purified using a high throughputprotein production process (HTPP). Selected binders were cloned into thepET9d vector in order to generate His₆ tag (SEQ ID NO: 487) fusions. DNAwas transformed into E. coli HMS174(DE3), and cells were inoculated in 5ml LB medium containing 50 μg/mL kanamycin in a 24-well format and grownat 37° C. overnight. Fresh 5 ml LB medium (50 μg/mL kanamycin) cultureswere prepared for inducible expression by aspirating 200 μl from theovernight culture and dispensing it into the appropriate well. Thecultures were grown at 37° C. until A₆₀₀ 0.6-0.9. After induction with 1mM isopropyl-β-thiogalactoside (IPTG), the culture was grown for another6 hours at 30° C. and harvested by centrifugation for 10 minutes at3220×g at 4° C. Cell pellets were frozen at 80° C.

Cell pellets (in 24-well format) were lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM imidazole, 1mg/ml lysozyme, 30 ug/ml DNAse, 2 μg/ml aprotonin, pH 8.0) and shaken atroom temperature for 1 hour. Lysates were clarified and re-racked into a96-well format by transfer into a 96-well Whatman GF/D Unifilter fittedwith a 96-well, 650 μl catch plate and centrifuged for 5 minutes at200×g. The clarified lysates were transferred to a 96-well Ni-ChelatingPlate that had been equilibrated with equilibration buffer (50 mMNaH₂PO₄, 0.5 M NaCl, 10 mM CHAPS, 40 mM imidazole, pH 8.0) and incubatedfor 5 minutes. Unbound material was removed by vacuum. The resin waswashed 2×0.3 ml/well with Wash buffer #1 (50 mM NaH₂PO₄, 0.5 M NaCl, 5mM CHAPS, 40 mM imidazole, pH 8.0) with each wash removed by vacuum.Next, the resin was washed with 3×0.3 ml/well with PBS with each washstep removed by vacuum. Prior to elution, each well was washed with 50μl Elution buffer (PBS+20 mM EDTA), incubated for 5 minutes, and thewash discarded by vacuum. Protein was eluted by applying an additional100 μl of Elution buffer to each well. After a 30 minute incubation atroom temperature, the plate(s) were centrifuged for 5 minutes at 200×gand eluted protein collected in 96-well catch plates containing 5 μl of0.5 M MgCl₂ affixed to the bottom of the Ni-plates. Eluted protein wasquantified using a BCA Protein assay with SEQ ID NO: 2 as the proteinstandard.

HTPP yielded active ¹⁰Fn3-based binders that were expressed in a solubleform and purified from the soluble fraction of the bacterial cytosol.FIG. 1 depicts an exemplary SDS-PAGE analysis from one of the E/I¹⁰Fn3-based binders. SEC analysis on a Superdex 200 5/150 GL in a mobilephase of 100 mM NaPO₄, 100 mM NaSO₄, 150 mM NaCl, pH 6.8 (GE Healthcare)demonstrated predominantly monomeric proteins (see Example 4).

In addition, midscale expression and purification of select ¹⁰Fn3-basedbinders was performed. The selected binders, fused to a His₆ tag (SEQ IDNO: 487), were cloned into a pET9d or pET29 vector and expressed in E.coli HMS174(DE3) or BL212(DE3) (EMD Biosciences, San Diego, Calif.)cells. 20 ml of an inoculum culture (generated from a single platedcolony) was used to inoculate 1 liter of LB medium containing 50 μg/mLkanamycin. The culture was grown at 37° C. until A₆₀₀ 0.6-1.0. Afterinduction with 1 mM isopropyl-β-thiogalactoside (IPTG), the culture wasgrown for another 6 hours at 30° C. Alternatively, expression wascarried out at 18° C. after initial growth at 37° C. using autoinductionmedia (“ONE” medium, EMD Biosciences, San Diego, Calif.). Cell pelletswere harvested by centrifugation for 30 minutes at ≧10,000×g at 4° C.and frozen at 80° C. The cell pellet was resuspended in 25 mL of lysisbuffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche), pH 7.4) using an Ultra-turrax homgenizer onice. Cell lysis was achieved by high pressure homogenization (≧18,000psi) using a Model M-110S Microfluidizer (Microfluidics). The solublefraction was separated by centrifugation for 30 minutes at 23,300×g at4° C. The supernatant was clarified via 0.45 μm filter. The clarifiedlysate was loaded onto a HisTrap column (GE) pre-equilibrated with 20 mMNaH₂PO₄, 0.5 M NaCl, pH 7.4. The column was then washed with 25 columnvolumes of 20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4, followed by 20 columnvolumes of 20 mM NaH₂PO₄, 0.5 M NaCl, 25 mM imidazole, pH 7.4, and then35 column volumes of 20 mM NaH₂PO₄, 0.5 M NaCl, 40 mM imidazole, pH 7.4.Protein was eluted with 15 column volumes of 20 mM NaH₂PO₄, 0.5 M NaCl,500 mM imidazole, pH 7.4, fractions pooled based on absorbance at A₂₈₀and dialyzed against 1×PBS, 50 mM Tris, 150 mM NaCl, pH 8.5 or 50 mMNaOAc, 150 mM NaCl, pH4.5. Any precipitate was removed by filtering at0.22 μm.

Midscale expression and purification yielded highly pure and activeproteins that were expressed in a soluble form and purified from thesoluble fraction of the bacterial cytosol. SEC analysis on a Superdex200 10/30GL in a mobile phase of 100 mM NaPO₄, 100 mM NaSO₄, 150 mMNaCl, pH 6.8 (GE Healthcare) demonstrated predominantly monomericproteins (see Example 4).

Example 3 Pegylation of E/I ¹⁰Fn3-Based Binders

Multi-valent fibronectin based scaffold proteins, such as E/I¹⁰Fn3-based binders, can be pegylated with various sizes and types ofPEG. To allow for pegylation, the protein is typically modified near theC-terminus by a single point mutation of an amino acid, typically aserine, to a cysteine. PEGylation of the protein at the single cysteineresidue is accomplished through conjugation with variousmaleimide-derivatized PEG forms by combining the derivitized-PEG reagentwith the protein solution and incubating. Progress and confirmation ofthe PEGylation conjugation reaction can be confirmed by SDS-PAGE and/orSE-HPLC methods that separate the non-PEGylated protein from thePEGylated protein.

For example, the construct E2-GS10-I1 (SEQ ID NO: 25) was pegylated byreplacing a serine that was at position 221 with a cysteine. Theresulting construct, SEQ ID NO: 56, was then conjugated with amaleimide-derivatized 40 kDa branched PEG (NOF America Corporation,White Plains, N.Y.). The derivatized PEG reagent was mixed with theprotein construct in solution and incubated at pH 7.40 at Roomtemperature until the reaction was complete, typically 30 minutes orovernight at 4° C. The pH was lowered to pH 4.5 or pH 5.0 by dialysis orrapid desalting using size exclusion column chromotography into in 50NaOAc, 150 mM NaCl buffer. The mixture of products and excess reactantsfrom the PEGylation reaction were then loaded onto a cation exchangechromotography column at the lowered pH and eluted with a 150 mM to 1 MNaCl gradient. Studies to confirm the pegylation were also conducted asdescribed in the paragraph above. The conjugations can be performed withthe His tagged or the His-Tag free versions of the protein.

On occasions in which E. coli endotoxin contamination needed to bedepleted in the sample, two methods used either separately or inconjunction with one another were employed. The first was to wash thecation exchange column with typically 5 column volumes NaOAc buffersupplemented with 0.5% Triton X-100, followed by 20 column volumes (ormore) of the same buffer without Triton X-100. Additionally or in placeof this procedure, the protein was passed very slowly through aSartorius Sartobind® Q filter (Sartorius Stedim Biotech Bohemia, N.Y.).

Two of the E/I ¹⁰Fn3-based binders, E2-GS10-I1-cys (with his) (SEQ IDNO: 56) and E3-GS10-I1-Cys (with his) (SEQ ID NO: 53), were pegylatedusing an alternative procedure. Five ml of an inoculum culture ofBL21(DE3) E. coli cells containing a T7 ploymerase driven pET29 plasmidencoding either E2-GS10-I1-cys (with his) or E3-GS10-I1-Cys (with his),were generated from a single plated colony and used to inoculate 1 literof auto-induction media (“ONE” medium, EMD Biosciences, San Diego,Calif.) containing 50 μg/mL kanamycin. Expression was carried out at 18°C. after initial growth at 37° C. and harvested by centrifugation for 10minutes at ˜10,000×g at 4° C. Cell pellets were frozen at 80° C. Thecell pellet was resuspended in 10 mL of lysis buffer (20 mM NaH₂PO₄, 0.5M NaCl, 5 mM Immidazole, pH 7.4) and mechanically lysed using an Avestinhomgenizer. The soluble fraction was separated by centrifugation for 15minutes at 23,300×g at 4° C. The supernatant was decanted and the pelletwas solubilized in Lysis buffer (above) supplemented with 4 M to 6 Mguanidine hydrochloride (GdnHCl). Solubilized protein was then purifiedon a suitably sized NiNTA column (Qiagen, Inc.) pre-equilibrated withthe GdnHCL supplemented Lysis Buffer. The column was then washed with 5to 10 column volumes of the same buffer, followed by elution with thesame buffer supplemented with 300 mM Immidazole. The fractions elutedoff the column containing the protein of interest were diluted to 2-3mgs/mL protein and then combined with a 1.2-1.5 molar excess of solidNEM-PEG (40 kDa branched or other). The mixture was allowed to react atroom temperature for 30 minutes or until the reaction was complete. Theentire reaction volume was then placed into a dialysis bag (5,000 DaMolecular Weight cutoff) and the mixture was subjected to a dialysisrefolding process. For example, this process may consist of two 10-16hour 500:1 (buffer:dialysate) dialysis exchanges against 50 mM NaOAc,150 mm NaCl, pH 4.5. The dialysate from this procedure contains properlyfolded, PEGylated materials plus excess reactants. The mixture ofproducts and excess reactants from the PEGylation reaction wereclarified via centrifugation or filtration prior to loading them onto acation exchange chromotography column (SP Sepharose or Resource S, GEHealthcare). The column was developed with 150 mM to 1 M NaCl gradientin the NaOAc background buffer. Studies to confirm the pegylation wereconducted as described above.

Example 4 Biophysical Characterization of ¹⁰ Fn3-Based Binders

Standard size exclusion chromatography (SEC) was performed on theproteins purified from the HTPP and the midscale processes (0.1 to 1 μgof protein for HTPP and 10-50 ug for midscale). SEC of HTPP derivedmaterial was performed using a Superdex 200 5/150 column (GE Healthcare)or on a Superdex 200 10/30 column (GE Healthcare) for midscaled materialon an Agilent 1100 or 1200 HPLC system with UV detection at A₂₁₄ nm andA₂₈₀ nm and with fluorescence detection (excitation=₂₈₀ nm, emission=₃₅₀nm). A buffer of 100 mM sodium sulfate, 100 mM sodium phosphate, 150 mMsodium chloride, pH 6.8 at appropriate flow rate of the SEC columnemployed. Gel filtration standards (Bio-Rad Laboratories, Hercules,Calif.) were used for molecular weight calibration.

The results of the SEC on the HTPP purified ¹⁰Fn3-based binders showedpredominantly monomeric proteins and elution in the approximate range of25 kDa vs. globular Gel Filtration standards (BioRad).

The results of the SEC on the midscaled purified ¹⁰Fn3-based bindersshowed predominantly monomeric proteins and elution in the approximaterange of 25 kDa vs. globular Gel Filtration standards (BioRad). FIG. 2depicts exemplary SEC profiles for E/I ¹⁰Fn3-based binders (I1-GS10-E2in FIGS. 2A and E2-GS10-I1 in FIG. 2B).

Select midscale ¹⁰Fn3-based binders were further analyzed by LC-MS(Water's 2695 liquid chromatography HPLC system coupled with WatersQ-TOF API mass spectrometer, Waters Corporation, Milford, Mass.).Samples were diluted to approximately 0.5 mg/ml with HPLC grade water.Approximately 5 μl of diluted sample was injected onto a Jupiter C18column (Catalog number 00G-4053-80, Phenomenex). Buffer A: 0.02%TFA+0.08% formic acid in HPLC grade water. Buffer B: 0.02% TFA+0.08%formic acid in HPLC grade acetonitrile. Sample was eluted with gradient(Table 1) at flow rate 0.2 ml/minutes.

TABLE 1 Time % A B % 0 95 5 5.00 75 25 25.00 55 45 30.00 5 95 32.00 95 545.00 95 5

HPLC elution was split at approximately to 1:1 ratio and half sent to UVdetector and the other half to mass spectrometer. Mass spectrometer hadthe following instrument settings: capillary voltage 3.5 KV, conevoltage 40, source temperature 80° C., desolvation temperature 250° C.,desolvation gas flow 450 and multi channel photo detector voltage 2200.Raw spectra were deconvoluted with MaxEn1 (Waters Corporation).

The molecular weight of I1-GS10-E2 (SEQ ID NO: 22) as measured by LC-MSis 24,445 Dalton, which is within 1 Dalton from the molecular weightcalculated from the amino acid composition. This indicates that theprotein has the correct amino acid composition and the N terminalmethionine is processed. There is no other post translationalmodification on the protein.

Differential Scanning calorimetry (DSC) analysis of the midscaledI1-GS10-E2 was performed to determine the T_(m). A 1 mg/ml solution wasscanned in a N-DSC II calorimeter (calorimetry Sciences Corp) by rampingthe temperature from 5° C. to 95° C. at a rate of 1 degree per minuteunder 3 atm pressure. The data was analyzed versus a control run of theappropriate buffer using a best fit using Orgin Software (OrginLabCorp). The results of this assay demonstrate that the E/I binder has aT_(m) of 50.69° C. (see FIG. 3A). Using the same methods, the T_(m) ofE2-GS10-I1 (with Peg) was determined to be 50.72° C. and the T_(m) ofE2-GS10-I1 (without Peg) was determined to be 56.82° C. (see FIG. 3B).

Example 5 Determination of Binding Affinity

Surface plasmon resonance (BIAcore) analysis was performed onsolution-phase ¹⁰Fn3-based binders in order to determine off-ratesand/or binding affinities using captured EGFR-Fc and IGF1R-Fc. Theextracellular domain of human IGF1R (aa 1-932) was cloned into amammalian expression vector containing the hinge and constant regions ofhuman IgG1. Transient transfection of the plasmid produced a fusionprotein, IGF1R-Fc which was subsequently purified by Protein Achromatography. Recombinant human EGFR-Fc (aa 1-645 of the extracellulardomain of human EGFR fused to human Fc) was purchased from R&D systems(Minneapolis, Minn.). IGF1R-Fc was captured on immobilized Protein Awhereas EGFR-Fc was captured on immobilized anti-human antibody.

In a typical experiment, anti-human IgG was immobilized on flow cells 1and 2 of a CM5 chip following the manufacturer's recommendations (GEHealthcare, Piscataway, N.J.). EGFR-Fc (50 nM) was injected at 5 uL for2 minutes on flow cell 2 (Fc2). Two 30 second injections of 3 M MgCl₂were used for regeneration of the bound EGFR-Fc from the anti-human IgGsurface. Protein A was diluted to 80 ug/mL in acetate pH 4.5 andimmobilized to ˜3000 RU on flow cells 3 and 4 of a CM5 chip surface.Approximately 1300 RU of IGF1R-Fc was captured on Fc 4. Two 30 secondinjections of 50 mM glycine pH 1.5 were used to regenerate the surfacebetween samples.

A concentration series of 100 nM to 1 nM of HTPP purified protein (threedata points collected) or 300 nM to 0.05 nM of midscale purified protein(eleven data points collected) was evaluated for binding to EGFR-Fc orIGF1R-Fc. Sensorgrams were obtained at each concentration and wereevaluated using Biacore T100 Evaluation Software, Version 1.1.1 (GEhealthcare/Biacore) to determine the rate constants k_(a) (k_(on)) andk_(d) (k_(off)). For the HTPP evaluation the off-rate was fitted fromthe 3 point curves. The affinity K_(D) was calculated from the ratio ofrate constants k_(off)/k_(on).

The EGFR ¹⁰Fn3-based binders were evaluated for specificity in a similarformat using anti-human IgG to capture HER2-Fc. The ¹⁰Fn3-based bindersdid not show any discernible binding to captured HER2-Fc underconditions where robust binding was seen for EGFR-Fc.

As shown in Table 2, both domains of the E/I ¹⁰Fn3-based binders arefunctional, retaining their binding properties to the respectivetargets. The off rates shown in Table 2 are from midscale material andare similar to the qualitative results obtained with the HTPP material.

TABLE 2 Summary of binding constants for ¹⁰Fn3-based binders TargetProtein ka (1/Ms) kd (1/s) K_(D) (nM) EGFR-Fc E1 1.19E+05 1.18E−03 9.921.43E+05 1.89E−03 13.2 E1-GS10-I1 6.29E+04 4.74E−04 7.53 3.82E+043.89E−04 10.17 I1-GS10-E1 1.26E+05 6.03E−04 4.8 4.13E+04 4.25E−04 10.28E2 3.73E+05 2.72E−04 0.73 3.27E+05  3.2E−04 0.98 E2-GS10-I1 3.93E+051.75E−04 0.45 3.75E+05 1.67E−04 0.45 I1-GS10-E2 6.47E+05 1.42E−04 0.223.90E+05 1.14E−04 0.29 E3 2.83E+05 3.98E−04 3.4 1.4 E3-GS10-I1 3.49E+052.29E−04 0.66 I1-GS10-E3 1.17E+05 2.91E−04 2.48 IGF1R-Fc I1 3.84E+064.34E−04 0.11 E1-GS10-I1 5.13E+05 3.38E−04 0.66 I1-GS10-E1 1.47E+063.98E−04 0.27 E2-GS10-I1 1.24E+06 3.95E−04 0.32 I1-GS10-E2 3.82E+064.79E−04 0.13 E3-GS10-I1  1.8E+06 2.09E−04 0.12 I1-GS10-E3 1.37E+064.54E−05 0.03

Example 6 Inhibition of IGFR activity in H292 Cells

The ability of E/I ¹⁰Fn3-based binders to inhibit phosphorylation ofIGF1R on tyrosine 1131 was determined using an H292 cell in vitro assay.Briefly, 65×10³ H292 cells were plated in 96-well microplates (BiocoatPoly-D-Lysine coated 96-well plate, cat#356640, Becton Dickinson,Franklin Lakes, N.J.) in RPMI-1640 culture medium containing 10 mM HepespH 7.4 and 10% fetal bovine serum. Cells were allowed to adhere for 24hours at 37° C., 5% CO₂. The next day cells were washed once with 200microliters per well of serum free RPMI-1640 and incubated overnight in100 μL per well of serum free RPMI-1640. Serial dilutions of HTPPmaterial was added and cells were incubated for an additional 3 hours.Cells were stimulated with 100 ng/ml of IGF-1 (cat#500-P11, PeproTech,Rocky Hill, N.J.) for 10 minutes at 37° C. Media was dumped from theplate and 100 μL of cell lysis buffer (Cell Signaling cat#9803, Beverly,Mass.) was added to each well. Cells were incubated at room temperaturefor 15 minutes to allow lysis and lysate was transferred to aphospho-IGFR ELISA (cat#7302, Cell Signaling, Beverly, Mass.). Themanufacturer's procedure was followed to carry out the ELISA.

As demonstrated in FIG. 4, His tagged E1-GS10-I1 inhibitedIGF1-stimulated phosphorylation of the IGF1R (IC₅₀=0.004 uM) withcomparable potency to the isolated IGF1R binder, I1 (IC₅₀=0.018 uM). TheEGFR binder, E1, alone had very little effect on IGF1R phosphorylation(IC₅₀>3.5 uM). As shown in FIG. 9, additional E/I binders demonstratedability to inhibit IGF1R-stimulated phosphorylation with an IC50 in therange of 0.1 nM to 19 nM, including several pegylated E/I binders thatwere tested. In particular, for the pegylated E/I binders E1-GS10-I1,and I1-GS10-E1, inhibition of pIGFR was shown at 0.9 nM and 4 nM,respectively. For pegylated E/I binders E2-GS10-I1 and I1-GS10-E2,inhibition of pIGFR was shown at 0.3 nM and 0.8 nM, respectively.

Example 7 Inhibition of EGFR Activity in H292 Cells

The ability of E/I ¹⁰Fn3-based binders to inhibit phosphorylation of theEGFR on tyrosine 1068 was determined using an H292 cell in vitro assay.The assay was carried out as described in Example 6, except that cellswere stimulated with 100 ng/ml of EGF (cat#236-EG-200, R & D Systems,Minneapolis, Minn.) and a phospho-EGFR ELISA was performed (cat#7240,Cell Signaling, Beverly, Mass.). The manufacturer's procedure wasfollowed to carry out the ELISA.

As demonstrated in FIG. 5, His-tagged E1-GS10-I1 inhibitedEGF-stimulated phosphorylation of the EGFR (IC₅₀=0.020 uM) withcomparable potency to the isolated EGFR binder, E1 (IC₅₀=0.007 uM). TheIGF1R binder, I1 alone had very little effect on EGFR phosphorylation(IC₅₀>6.21 uM). As shown in FIG. 9, additional E/I binders demonstratedability to inhibit EGF-stimulated phosphorylation with an IC50 in therange of 7 nM to 127 nM, including several pegylated E/I binders thatwere tested. In particular, for pegylated E2-GS10-I1 and I1-GS10-E2,inhibition of pEGFR was shown at 32 nM and 47 nM, respectively. Similardata is shown in FIG. 11 for the pegylated E/I binders E2-GS10-I1, andI1-GS10-E2.

Example 8 Inhibition of EGF+IGF1-Induced pAKT in H292 Cells

The ability of E/I ¹⁰Fn3-based binders to inhibit phosphorylation of AKTon serine 473 was determined using an H292 cell in vitro assay. Theassay was carried out as described in Example 6, except that cells weresimultaneously stimulated with both EGF and IGF1 as described above andlysates were analyzed with a phospho-AKT ELISA (cat#7160, CellSignaling, Beverly, Mass.). The manufacturer's procedure was followed tocarry out the ELISA.

Signal transduction at EGFR and IGF1R feeds into the PI3K-AKT signalingpathway and stimulates phosphorylation of AKT. As demonstrated in FIG.6, E1-GS10-I1 inhibited EGF and IGF1-stimulated phosphorylation of AKTin H292 cells. The E/I ¹⁰Fn3-based binder was slightly more potent inits ability to block AKT activation (IC₅₀=0.004 uM) than the IGF1Rbinder, I1, by itself (IC₅₀=0.031 uM). The EGFR binder, E1, exhibitedonly modest activity in its ability to block AKT activation by bothligands (IC₅₀=1.28 uM). As shown in FIG. 9, additional E/I bindersdemonstrated ability to inhibit EGF and IGF1-stimulated phosphorylationof AKT with an IC50 in the range of 0.1 nM to 26 nM, including severalpegylated E/I binders that were tested.

Example 9 Inhibition of Cell Proliferation in RH41 and H292 Cells

E/I ¹⁰Fn3-based binders were evaluated for antiproliferative activity inthe H292 non-small cell lung carcinoma cell line, which depends on EGFRsignaling for growth, or the RH41 Ewing sarcoma cell line, which dependson IGF1R signaling for growth. Antiproliferative activity of binders wasassessed in monolayer cultures by staining cellular DNA with theCyQuantNF fluorescent stain (cat#C35006, Invitrogen, Carlsbad, Calif.).Briefly, 2×10³ H292 or 5×10³ RH41 cells were plated into 96-wellmicroplates (View Plates 96F cat#6005225, Perkin-Elmer, Waltham, Mass.)in RPMI-1640 culture medium containing 10 mM Hepes pH 7.4 and 10% fetalbovine serum and allowed to adhere for 24 hours at 37° C., 5% CO₂. Cellswere maintained as exponentially growing monolayers and remained inlogarithmic growth phase during the period of the assay without reachingconfluence during the course of the assay. Twenty-four hours afterplating, serial dilutions of midscale material was added and cells wereincubated for an additional 72 hours. Following this incubation, cellswere treated with CyQuantNF reagent and allowed to incorporate dye intocellular DNA for 1 hour at 37° C. Total DNA was quantified by readingfluorescence at 485 nm excitation and 530 nm emission on a CytoFluor4000 instrument (Applied Biosystems, Framingham, Mass.). Total time thatcells were exposed to drug was 72 hours. Standard compounds wereincluded in each experiment to verify assay performance andreproducibility. Linear regression analysis of the percent of inhibitionby test compound was used to determine IC₅₀ values.

As demonstrated in FIG. 7, in RH41 cells, His-tagged E1-GS10-I1inhibited proliferation with comparable potency (IC₅₀=0.009 uM) to theIGFR binder, I1 (IC₅₀=0.028 uM). The EGFR binder, E1, by itself had verylittle effect on the proliferation in this cell line (IC₅₀>12.5 uM).

As demonstrated in FIG. 8, in H292 cells, His-tagged E2-GS10-I1inhibited proliferation with greater potency (IC₅₀=0.329 uM) than theIGFR binder, I1, (IC₅₀=0.699 uM) or the EGFR binder, E2 (IC₅₀=0.553 uM).See Table 4 below for the IC50 values for the E and I monomers.

Example 10 Competitive EGF Ligand Binding Assay

The E/I binders E1-GS10-I1, I1-GS10-E1, E2-GS10-I1 and I1-GS10-E2 (HTPPmaterial) were tested in an EGF ligand binding cell-based competitionassay in A431 cells and compared to EGFR ¹⁰Fn3-based binders E1 and E2(midscale material). A431 cells were plated at 15000 cells/well in96-well plates in DMEM+10% FBS and incubated 48 hours. Cells were washedwith starvation media (DMEM+0.1% BSA) and incubated in starvation mediafor 1 hour. Starvation media was removed and replaced with ¹⁰Fn3-basedbinders that were diluted in starvation media and cells werepre-incubated for 30 minutes at 37° C. to allow proteins to bind to EGFreceptors on cell surfaces. 10 nM final concentration of Europium(Eu)-labeled EGF (Perkin Elmer, Boston, Mass.) diluted in starvationmedia was added to pre-incubated cells and plates were incubated for 3hours at 4° C. in the dark. Plates were washed twice with cold PBS and50 ul/well of Enhancement solution (Perkin Elmer, Boston, Mass.) wasadded to plates and incubated 1 hour at 37° C. Plates were read on theFlexstation II (Molecular Devices). The data was plotted with Softmaxplus software and IC50 values, i.e., the concentration of ¹⁰Fn3-basedbinders required to inhibit 50% of the Eu-EGF ligand from binding to theEGF receptor on the cell surfaces, were calculated.

The results for E2 and E1 compared with E2-GS10-I1, 11-GS10-E2,E1-GS10-I1 and I1-GS10-E1 are summarized in Table 3. This data indicatesthat the E/I ¹⁰Fn3-based binders compete with, and inhibit the bindingof, EGF to the EGFR receptor on A431 cells with similar potency to theEGFR ¹⁰Fn3-based binders. See Table 4 below for the IC50 values for theE and I monomers.

TABLE 3 Summary of IC50 values for inhibition of EGF Binding to EGFR onA431 cell surfaces Protein IC50 (nM) E2 7 E1 14 E2-GS10-I1 1.8I1-GS10-E2 1.4 E1-GS10-I1 14.6 I1-GS10-E1 7

Example 11 Activation and Signaling Activity in Cell-Based Assays

Target effects of the various E/I ¹⁰Fn3-based binders were evaluated inDiFi colon carcinoma cells by immunoblotting. Cells were seeded at 4×10⁵cells in each 25 cm² flask and incubated overnight at 37° C. in 5% CO₂.The next day, treatments were initiated and cells were further incubatedfor various times from 1.5 to 120 hours. Cells were then lysed in HNTG(50 mM Hepes, 150 mM NaCl, 0.5% triton-X-100, 8% glycerol, 2 mM Na₃VO₄,1.5 mM MgCl₂, 1 mM EDTA containing the protease inhibitors AEBSF,aprotinin, leupeptin, bestatin, pepstatin-A and E64) and total proteinwas quantified with the BCA protein assay (Pierce, Waltham, Mass.).Levels of total EGFR, total IGF1R and the phosphorylation state of theEGFR, MAP kinase protein ERK1/2 isoforms, was detected by SDS-PAGEanalysis of 20 micrograms of total protein followed by transfer ofproteins to nitrocellulose and immunoblotting with specific antibodies.Blots were also probed with β-actin to demonstrate equal loading of eachsample.

The pegylated E/I ¹⁰Fn3-based binders E1-GS10-I1 (SEQ ID NO: 55),E2-GS10-I1 (SEQ ID NO: 56), and E3-GS10-I1 (SEQ ID NO: 53), demonstratedthe ability to degrade EGFR in this assay. In addition, for E3-GS10-I1(SEQ ID NO: 53), degradation of IGF1R was also observed. The effect onEGFR degradation for the pegylated binder E2-GS10-I1 is shown in FIG.10, as are other effects on signaling molecules. Additionally, thenon-pegylated version of the binder E2-GS10-I1 demonstrated similar EGFRdegradation (data not shown). FIG. 10 shows that for the pegylatedbinder I1-GS10-E2, there was no EGFR degradation. Table 4 belowsummarizes various properties of the E monomers.

TABLE 4 Summary of properties of E monomers. EGFR Neutralizes InhibitionInhibition Inhibition of BIAcore EGF of of H292 KD Binding pEGFR pERKProliferation Monomer IC50 IC50 IC50 IC50 IC50 E1 14.6 nM 0.53 nM 18 nM17 nM 18 nM E2  1.4 nM 1.46 nM 20 nM 40 nM 30 nM E3 0.72 nM 0.87 nM 11nM 97 nM 26 nM

Example 12 Evaluation of Certain E/I ¹⁰Fn3-Based Binders on H292 TumorXenografts Grown in Nude Mice

The pegylated E/I binders E2-GS10-I1 and E3-GS10-I1 as well as themonoclonal antibody panitumumab were evaluated in an H292 tumorxenograft model. For in vivo models, panitumumab was obtained as themarketed drug and E/I binders were purified as described above. In vitroactivity of all E/I binders was validated prior to administration inanimals by testing functionality of each end in the EGF-stimulated pEGFRand the IGF1-stimulated pIGFR assay in H292 cells. E/I binders werediluted in phosphate buffered saline (PBS) at the beginning of theexperiment and stored at 2-4° C. for the duration of each study. Bothcompounds were administered i.p. in a total volume of 500 μl/inj/mouseand were equilibrated to room temperature prior to administration.

Mice and Tumor Propagation.

Female athymic (nude) mice 5-6 weeks of age were obtained from HarlanSprague-Dawley Co. (Indianapolis, Ind.). and were quarantined forapproximately 3 weeks prior to their use for tumor propagation or drugefficacy testing. The animals were provided food and water ad libitum.Animal care was performed in keeping with AAALAC and Bristol-MyersSquibb guidelines. Tumors were propagated by subcutaneous (s.c.)implantation in nude mice. Tumor passages occurred approximately everytwo to four weeks.

In vivo antitumor testing. Estimated tumor weight was calculated usingthe formula: Tumor weight (mg)=(w²*l)/2; where w=width and l=length inmm. Antitumor activity was evaluated in terms of % tumor growthinhibition (TGI) where a % TGI of >50% was considered active. Relative %tumor growth inhibition was calculated as %TGI=[(C_(t)−T_(t))/(C_(t)−C₀)]×100 where C_(t)=median tumor weight ofcontrol mice at time t in days after tumor implant, T=median tumorweight of treated mice at time t, C₀=median tumor weight of control miceat time 0. % TGI value was calculated at various time points beginningafter 1.5 tumor volume doubling times and sustained over a time periodof 3 tumor volume doubling times (TVDT) where possible. Where,TVDT=median time (days) for control tumors to reach target size—mediantime (days) for control tumors to reach half the target size. Thedefinition of a cured mouse was one whose tumor was undetectable, or <35mg, when assessed more than 10 TVDTs post-treatment. The dose of acompound which yielded the maximum therapeutic effect, was termed theoptimal dose (OD). Treatment groups (typically 8 mice) with more thanone death attributable to drug toxicity were considered to have hadexcessively toxic treatments and their data were not used in theevaluation of antitumor activity. The maximum tolerated dose (MTD) isdefined as the dose level immediately below which excessive toxicity(i.e. more than one death) occurred. Treated mice dying prior to havingtheir tumors reach target size were considered to have died from drugtoxicity. Statistical evaluations of data were performed using Gehan'sgeneralized Wilcoxon test (Gehan, E A, A Generalized Wilcoxon Test forComparing Arbitrarily Slightly-Censored Samples, Biometrika 52:203-223,1965).

Measurement of Pharmacodynamic Endpoints in Tumors.

Tumors were harvested from untreated or drug treated mice and snapfrozen in liquid nitrogen. Samples were weighed and homogenized in 10 μlof lysis buffer (50 mM Hepes, 150 mM NaCl, 0.5% triton-X-100, 8%glycerol, 2 mM Na₃VO₄, 1.5 mM MgCl₂, 1 mM EDTA containing one completemini protease inhibitor tablet Sigma #S 8820 per 15 ml buffer andphosphatase inhibitor cocktail Sigma #P5726) for each mg of tissue.Tissues were minced in a 100 mm petri dish with two scalpels,transferred to Falcon#2059 polypropylene round bottom tubes andmacerated with a hand held homogenizer for 30 seconds. Homogenate wastransferred to 1.5 ml eppendorf tubes and centrifuged at 15000×g for 2minutes in a microfuge. Clarified supernatant was transferred to a newtube and total protein concentration was determined with the Pierce BCAprotein assay (Pierce Biotechnology). Samples were analyzed byimmunoblotting or on a Meso scale MSD Sector Imager 6000 multi spotassay system as recommended by the manufacturer (Meso Scale Discovery,Gaithersburg, Md.).

The pegylated E/I binders E2-GS10-I1 and E3-GS10-I1 were tested in anH292 NSCLC in athymic mice. Tumors were implanted subcutaneously with 1mm³ H292 tumor fragments in the hind flank and allowed to establish to asize of 50-150 mg prior to initiation of treatment on Day 6 post-tumorimplant. The pegylated E/I binders were administered i.p. at a dose of100 mg/kg on a TIWX3 schedule to assess antitumor activity. Panitumumabwas obtained as marketed drug and administered i.p. at its optimal doseof 1 mg/mouse and at a lower dose of 0.1 mg/mouse on a Q3DX5 schedule.Mean tumor sizes calculated from groups of 8 mice are shown in FIG. 12A.The 1 mg/mouse and 0.1 mg/mouse doses of panitumumab were both active by% TGI with values of 101% and 100%, respectively and these values weresignificantly different from control animals (p=0.0002, Table 5).Pegylated E2-GS10-I1 was also significantly active by % TGI with a valueof 96% (p=0.0005). Pegylated E3-GS10-I1 was not active in this studywith a % TGI value of 31% that was not statistically different from thecontrol group (p=0.416). Post dosing analysis indicated thatapproximately two thirds of the pegylated E3-GS10-I1 was aggregated(66.64% aggregation/33.36% monomer for one batch and 72.53%aggregation/27.47% monomer for another batch) which could account forthe poor activity of pegylated E3-GS10-I1 in this assay. In contrast,the pegylated E2-GS10-I1 showed only a small percentage of aggregationin post dosing studies (1.79% aggregation/98.21% monomer).

All treatments were well tolerated with no treatment related deaths orexcessive weight loss over the course of the study. Clinicalobservations revealed no evidence of toxicity and the average weightchange over the course of therapy was within acceptable limits (FIG.12B).

TABLE 5 Results of the H292 human tumor xenograft study Schedule, DoseAVE weight p value for Outcome Group Compound Route (mg/kg) change (g) %TGI % TGI by % TGI 1 Control (untreated) — — 5.3 — 1.0 — 2 panitumumabq3dx5; 6  1 mg/mse 9.6 101 0.0002 A ip^(a) 3 panitumumab q3dxp5; 6  0.1mg/mse 6.3 100 0.0002 A ip^(a) 4 E2-GS10-I1 (w/ PEG) TIWx3; 6 100 −0.194 0.0005 A ip^(a) 5 E3-GS10-I1 (w/ PEG) TIWx3; 6 100 9.5 28 0.416 Iip^(a) ^(a)Vehicle was phosphate buffered saline. Abbreviations used areas follows: ip, intraperitoneal route; % TGI, relative % tumor growthinhibition calculated as % TGI = [(Ct − Tt)/(Ct − C0)] × 100 where Ct =median tumor weight of control mice at time t in days after tumorimplant, Tt = median tumor weight of treated mice at time t, C0 = mediantumor weight of control mice at time 0. % TGI value was calculated attwo points as the average inhibition of Day 12 and Day20. Outcome, atreatment regimen was considered active if it produced a statisticallysignificant % TGI value of >50%; q3dx5; 6, compound was administeredevery three days for five doses starting on the sixth day after tumorimplant; TIWx3; 6, compound was administered three times a week forthree weeks starting on the sixth day after tumor implant. p values werecalculated on Day 20 relative to the control group in a two tailedpaired analysis with 8 measurements per group. Outcome by % TGI, A =active and I = inactive.

Pharmacodynamic Endpoints from the H292 Tumor Study.

Samples of tumors from untreated control, panitumumab and E/I bindertreated groups were analyzed for levels of phosphorylated EGFR, ErbB2and IGFR that would indicate target suppression. Tumors were alsoanalyzed for levels of total EGFR to determine if EGF receptordegradation occurred. On day 20, a final treatment was administered andtumors were removed from 2 animals at 1 hour after dosing, 3 animals at4 hours after dosing and 3 animals at 24 hours after dosing. Alltreatments showed marked suppression of phosphorylated EGFR and ErbB2while the basal levels of phosphorylated IGFR were too low to discern adifference in this study (FIG. 13). All treatments showed a reduction inthe amount of total EGFR indicating degradation of the receptor hadoccurred.

Example 13 Selection and Characterization of MCF7 Cells Resistant toIGF1R Inhibitor

MCF7 cells (American Type Culture Collection, Cat No. HTB-22, Manassas,Va.) were cultured in RPMI medium containing 10 mM hepes and 10% FBS at37° C. in the presence of 5% CO₂. The small molecule IGF1R inhibitorBMS-754807 was added to the culture medium and the concentrationincreased at stepwise increments over a period of 10 months until thecells exhibited continued proliferation in the presence of 200 mMBMS-754807. The resistant cells were designated MCF7r and the IC50 forBMS-754807 was 1239 nM compared to 120 nM for the parental MCF7 cells asmeasured in a proliferation assay carried out as previously described(Carboni et al., Cancer Res. 69: 161-170 (2009)). The drug was thenremoved from the culture medium and the MCF7r cells were passaged incomplete medium for an additional 20 or 60 days to remove all traces ofresidual BMS-754807. Analysis of the MCF7r cells by immunoblottingrevealed that EGFR was significantly overexpressed in the resistantcells compared to the parental MCF7 cells (FIG. 14). In addition, whenMCF7 and MCF7r cells were serum starved and then stimulated with EGF for7 minutes, phosphorylated EGFR could not be detected in the parentalMCF7 cells (probably due to low levels of EGFR) but was strongly visiblein MCF7r cells. In serum starved cells stimulated with IGF ligand,phosphorylated IGFR was seen in the parental MCF7 cells but despite theslightly higher levels of total IGFR present in the MCF7r cells almostno pIGFR was observed. This shows that the IGFR in the resistant MCF7rcells lost the ability to activate IGFR in response to IGF1 stimulation(FIG. 14). Activation of the MAP kinase pathway in response to EGFstimulation was stronger in the MCF7r cells as measured by pERKactivation.

Example 14 Antitumor Studies in MCF7 and MCF7r Xenografts

MCF7r cells were scaled up in T75 flasks and isolated by centrifugation.Viable cell numbers were measured by trypan blue exclusion with aVi-CELL XR (Beckman Coulter, Fullerton, Calif.), resuspended in PBS to5×10⁶ viable cells/ml and implanted subcutaneously in the hind flank ofathymic mice in a volume of 0.2 ml. For MCF7 and MCF7r tumor growth, allmice were supplemented with 0.25 mg 90 day release pellets of17-β-estradiol (Innovative Research of America, Sarasota, Fla., Cat. No.NE-121). Tumors were propagated until they reached a median size of500-1000 mg when they were excised and 1 mm³ fragments were reimplantedin the hind flank of new athymic mice. Tumors were adapted for solidtumor growth by serial trocar passage in mice through at least fourrounds of growth during which tumor volume doubling time and take ratewere monitored for each passage. Growth characteristics were observed todetermine if the xenografts exhibited acceptable properties to serve asa reliable, reproducible model. The MCF7r tumor type demonstrated anacceptable take rate and doubling time and therefore satisfied thecriteria for use as a xenograft model. The MCF7 parental tumor model hadbeen previously established using the same techniques. For the MCF7parental xenograft, 1 mm³ tumor fragments were implanted subcutaneouslyin the hind flank and allowed to establish to a size of 50-150 mg priorto initiation of treatment on Day 13 post-tumor implant. Cetuximab wasobtained as marketed drug and administered i.p. at its optimal dose of 1mg/mouse and at a lower dose of 0.1 mg/mouse on a Q3DX5 schedule (dosesadministered on Day 13, 16, 19, 22, 25). Mean tumor sizes calculatedfrom groups of 8 mice are shown in FIG. 15A. In the MCF7 xenograftmodel, neither the 1 mg/mouse or the 0.1 mg/mouse dose of cetuximab wasactive by % TGI with values of −9% and 3.2%, respectively and the tumorsizes were not statistically different from the control group (Table 6).

For the MCF7r resistant xenograft, 1 mm³ tumor fragments were implantedsubcutaneously in the hind flank and allowed to establish to a size of50-150 mg prior to initiation of treatment on Day 6 post-tumor implant.Cetuximab was obtained as marketed drug and administered i.p. at itsoptimal dose of 1 mg/mouse and at a lower dose of 0.1 mg/mouse on aQ3DX5 schedule (doses administered on Day 6, 9, 12, 15, 18). Mean tumorsizes calculated from groups of 8 mice are shown in FIG. 15B. In theMCF7r xenograft model, doses of cetuximab were active by % TGI withvalues of 105% and 75%, respectively. The high dose of cetuximab had aTGI value over 100% which indicates that it caused tumor regressionbelow the starting size at the initiation of treatment. Both dosesresulted in a statistically significant difference in tumor sizecompared to the control group (Table 7).

TABLE 6 Results of the MCF7 human breast carcinoma tumor xenograftstudy. Schedule, Dose p value for Outcome Group Compound Route(mg/mouse) % TGI % TGI by % TGI 1 Control (untreated) — — — 1.0 — 2cetuximab q3dx5; 13 ip^(a)   1 mg/mse −9 0.223 I 3 cetuximab q3dx5; 13ip^(a) 0.1 mg/mse 3.2 0.220 I ^(a)Vehicle was phosphate buffered saline.Abbreviations used are as follows: ip, intraperitoneal route; % TGI,relative % tumor growth inhibition calculated as % TGI = [(Ct − Tt)/(Ct− C0)] × 100 where Ct = median tumor weight of control mice at time t indays after tumor implant, Tt = median tumor weight of treated mice attime t, C0 = median tumor weight of control mice at time 0. % TGI valuewas calculated at three points as the average inhibition of Day 20, 24and Day 27. Outcome, a treatment regimen was considered active if itproduced a statistically significant % TGI value of >50%; q3dx5; 13,compound was administered every three days for six doses starting on thethirteenth day after tumor implant. p values were calculated on Day 24relative to the control group in a two tailed paired analysis with 8measurements per group. Outcome by % TGI, A = active and I = inactive.

TABLE 7 Results of the MCF7r human breast carcinoma tumor xenograftstudy. Schedule, Dose p value for Outcome Group Compound Route (mg/kg) %TGI % TGI by % TGI 1 Control (untreated) — — — 1.0 — 2 cetuximab q3dx5;6 ip^(a)   1 mg/mse 105 0.001 A 3 cetuximab q3dx5; 6 ip^(a) 0.1 mg/mse 75 0.024 A See footnotes to Table 6. p values were calculated on Day 19relative to the control group in a two tailed paired analysis with 8measurements per group.

Example 15 Antitumor Studies in GEO Xenografts

GEO tumors were established by implanting 1 mm³ tumor fragmentssubcutaneously in the hind flank of athymic mice and allowing them toreach a size of 50-150 mg prior to initiation of treatment on Day 18post-tumor implant. Cetuximab was administered ip at 0.25 mg/mouse on aQ3DX5 schedule (doses administered on Day 18, 21, 24, 27, 30). The IGFRkinase inhibitor BMS-754807 was administered at 25 mg/kg on a QDX21schedule. Mean tumor sizes calculated from groups of 8 mice are shown inFIG. 16. Cetuximab was active at 0.25 mg/mouse with a % TGI value of67%. BMS-754807 was active with a % TGI of 80% and the combination ofthe two was considerably more active then either agent alone with a %TGI of 94% (Table 8). All treatment groups were statistically differentfrom the control group on Day 26 (Table 8).

TABLE 8 Results of the GEO human colon carcinoma tumor xenograft study.Cetuximab Dose BMS-754807 Schedule, (mg/ Schedule, Dose p Outcome GroupRoute mouse) Route (mg/kg) % TGI value by % TGI Synergy 1 Control — — —— — — — (untreated) 2 q3dx5; 6 ip^(a) 0.25 mg/ — 80 A — mse 3 — — qdx21;18^(b) 25 67 A — 4 q3dx5; 6 ip^(a) 0.25 mg/ qdx21; 18^(b) 25 94 A YESmse ^(a)Vehicle for cetuximab was phosphate buffered saline. Vehicle forBMS-754807 was 50% polyethylene glycol 400, 50% water. Abbreviationsused are as described in Table 6 and synergy is defined as statisticallysignificant activity that is better than either agent in the combinationdemonstrated on its own. Outcome by % TGI, A = active and I = inactive.

Example 16 Antitumor Studies in H292 Xenografts

H292 cells were implanted subcutaneously in the hind flank of athymicmice as 1 mm³ fragments and allowed to establish to a size of 50-150 mgprior to initiation of treatment on Day 12 post-tumor implant. Cetuximabwas administered ip at 0.1 mg/mouse on a Q3DX5 schedule. MAB391 is anantibody to IGF1R(R&D Systems, Minneapolis, Minn., Cat. No. MAB391) andwas administered at a dose of 40 mg/kg on a BIWX3 schedule. Mean tumorsizes calculated from groups of 8 mice are shown in FIG. 17. Cetuximabwas active at 0.1 mg/mouse with a % TGI value of 95.1% and MAB391 wasinactive at 40 mg/kg with a % TGI value of 10.5% (Table 9). Mice dosedwith the combination of cetuximab and MAB391 exhibited a % TGI value of109.2% indicating tumor regression in the combination group (Table 9).After dosing ceased, tumors regrew in the cetuximab treated group morerapidly than in the group treated with the combination of cetuximab andMAB391 (FIG. 17).

TABLE 9 Results of the H292 human NSCLC tumor xenograft study. CetuximabDose MAB391 Schedule, (mg/ Schedule, Dose p Outcome Group Route mouse)Route (mg/kg) % TGI value by % TGI Synergy 1 Control — — — — — — —(untreated) 2 q3dx5; 12 ip^(a) 0.1 mg/ — 95.1 A — mse 3 — — BIWx3;12^(a) 40 10.5 I — 4 q3dx5; 12 ip^(a) 0.1 mg/ BIWx3; 12^(a) 40 109.2  AYES mse ^(a)Vehicle for cetuximab and MAB391 was phosphate bufferedsaline. Abbreviations used are as described in Table 6 and 8.

Example 17 Colony Formation Assay

To determine the effects of test compounds on the ability to inhibitcolony formation of H292 cells, 400 cells were seeded into 24-wellplates (Becton-Dickinson, Franklin Lakes, N.J., Cat. No. 351143) incomplete medium and allowed to adhere overnight. The next day medium wasremoved and replaced with medium containing 2% FBS. Test compound wasdiluted into medium containing 2% FBS and added to cells in serialdilutions. Cells were incubated at 37° C. for 14 days. After 14 days,media was discarded and wells rinsed once with 2 ml PBS. Cells werestained with 0.5 ml Coomassie Stain Solution (Bio-Rad, Hercules, Calif.,Cat. No. 161-0436) for 20 min. The stain was aspirated and wells werewashed quickly with 1× Destain Solution Coomassie R-250 (Bio-Rad, Cat.No. 161-0438). A final rinse with 1 ml water per well was carried outand plates were inverted and allowed to dry. Colonies consisting of (atleast) 50 cells or larger were counted by eye under low powermagnification (10×-20×). All samples were tested in triplicate and IC50values were calculated from linear regression of the percent inhibitionof control. Representative results for a PEGylated E/I binder are shownin FIG. 18 and IC50 values for various E/I ¹⁰Fn3-based binders,monospecific IGF1R ¹⁰Fn3-based binder, and EGFR antibody is shown inTable 10.

TABLE 10 IC50 values of various E/I ¹⁰Fn3-based binders, monospecificIGF1R ¹⁰Fn3-based binder, and EGFR antibody in the colony formationassay. IC50 SAMPLE (nM) E4-GS10-I1 (with Peg) 5 I1-GS10-E5 (with Peg) 1I1-GS10-E4 6 E2-GS10-I1 (with Peg) 560 I1 monomer (with Peg) 15.510panitumumab 140

Example 18 Epitope Mapping Assay

An epitope mapping assay was developed utilizing commercially availableantibodies where the binding site on the EGFR extracellular domain isroughly known according to various literature reports. The antibodiesused in this assay are listed in Table 11 and FIG. 19A depicts howantibodies were localized to approximate binding domains on EGFR. Theassay is a variation of the In Cell Western assay previously describedand assesses the ability of EGFR ¹⁰Fn3-based binders preincubated withA431 or other cells expressing EGFR to block binding of the detectionantibodies from the panel listed in Table 11. The assay was carried outas follows: A431 cells in log phase growth were harvested bytrypsinization and seeded in a 96 well plate at 24,000 cells/well in atotal volume of 100 μl/well. The next day, media was dumped and the EGFR¹⁰Fn3-based binders diluted in cold DMEM base media were added to theplate and allowed to bind for 1 hour at 4° C. to prevent internalizationof EGFR. After binding, cells were washed with 0.2 ml PBS+0.1% Tween-20and fixed for 20 minutes in PBS+3.7% formaldehyde at room temp. Cellswere blocked in 0.2 ml of Odyssey blocking buffer for 1 hour at roomtemp. Next, primary antibodies were diluted in 50 μl of Odyssey blockerper well and incubated for 1-2 hours at room temp. Primary antibodieswere dumped by inverting the plate, and each well washed 3× with 200 μlof PBS+0.1% Tween-20. Secondary antibodies are the same ones used in theIn Cell Western assay and were appropriate for the species of antibodybeing detected. These secondary antibodies were diluted (1:800) inOdyssey Blocker+0.2% Tween-20 and added in a volume of 50 μl per wellalong with TOPRO3 (Invitrogen, Carlsbad, Calif., cat#T3605) diluted at(1:3000) to counterstain cells for normalization. Cells were incubatedon bench for 1 hour at room temp. Secondary antibody was dumped out andeach well washed 4× with 200 μl of PBS+0.1% Tween-20 for 5 minutes atroom temp. Plates were imaged on a Licor instrument at 160 μmresolution, medium quality, focus offset of 3 mm, intensity of 5. Thisassay was also carried out with the marketed drug antibodies cetuximab,panitumumab and nimotuzumab to determine if the EGFR ¹⁰Fn3-based binderswere interfering with their binding to EGFR on A431 cells.Representative results are shown in FIG. 19B.

TABLE 11 Commercially available antibodies to the extracellular domainof EGFR. Binding Clone SUPPLIER and cat# SPECIES BINDS EPITOPE motif 1Abcam ab38165 Rab h Peptide AA 42-58 linear 2 E234 Abcam ab32198 Rab h,mu, rat Peptide AA 40-80 (No ICC) linear 3 N-20 Santa Cruz#31155 GoatIgG h AA 110-160 linear 4 ICR10 Abcam ab231 Rat IgG2a h(HN5) AA124-176^(b), neutralizing^(e) conf Santa Cruz #57095 5 EGFR1 Abcam ab30Mu IgG1 h(A431) AA 176-294, neutralizing^(b) conf Chemicon MAB88910ab30&MAB88910@(1 mg/ml) Labvision MS-311 6 199.12 Labvision MS-396-P MuIgG2a h AA 124-176, non-neutralizing^(b) conf 7 LA22 Upstate 05-104 MuIgG2a h(A431) AA 351-364, neutralizing^(a) linear 8 Abcam ab15669 RabMu, rat Peptide AA376-394^(d) linear 9 225 Sigma E2156 Mu IgG1 h(A431)AA 294-475, neutralizing^(b,c) conf Labvision MS-269-P 10 528 Abcamab3103 Mu IgG2a h(A431) AA 294-475, neutralizing^(b,c) conf SantaCruz#120 Labvision MS-268-P 11 B1D8 Labvision MS-666-P Mu IgG2a h(A431)AA 294-475^(b) conf 12 LA1 Upstate 05-101 Mu IgG1 h neutralizing 13 H11Labvision MS-316-P Mu IgG1 h AA 294-475, non-neutralizing^(b) linear 14111.6 Labvision MS-378-P Mu IgG1 h AA 294-475, neutralizing^(b) linearImgenex IMG-80179 15  29.1 Sigma E2760 Mu IgG1 h(A431) Externalcarbohydrate non- Abcam ab10414 neutralizing Abbreviations: conf:epitope conformationally specific; linear: epitope independent ofconformation. ^(a)JBC 264(1989)17469 Ala351-Asp364, ^(b)J ImmunologicalMethods 287(2004)147, ^(c)Mol Biol Med1(1983)511, ^(d)Raised against apeptide to mouse EGFR [FKGDSFTRTPPLDPRELEI (SEQ ID NO: 491)], ^(e)Int JOncol 4(1994)277.^(f)[EEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNY(SEQ ID NO: 492)], ^(g)Ile-Gln-Cys-Ala-His-Tyr-Ile-Asp-Gly-Pro-His-Cys(SEQ ID NO: 493) (amino acids 580-591). ^(h)Cancer Cell 7(2005)301.

Using various approaches, we have confirmed that the EGFR monomer E3binds to domain I of EGFR. Since other E monomers have similarproperties in various experiments, it is thought that the other Emonomers also bind to domain I of EGFR.

Example 19 Properties of I Monomers

BIAcore Analysis of the Soluble Fibronectin-Based Scaffold Proteins

The kinetics of I monomers binding to the target was measured usingBIAcore 2000 or 3000 biosensors (Pharmacia Biosensor). A capture assaywas developed utilizing an IGF-1R-Fc fusion. A similar reagent had beendescribed by Forbes et al. (Forbes et al. 2002, European J.Biochemistry, 269, 961-968). The extracellular domain of human IGF-IR(aa 1-932) was cloned into a mammalian expression vector containing thehinge and constant regions of human IgG1. Transient transfection of theplasmid produced a fusion protein, IGF-1R-Fc which was subsequentlypurified by Protein A chromatography and captured on Protein Aimmobilized on Biasensor CM5 chips by amine coupling. The kineticanalysis involved the capture of IGF-1R-Fc on Protein A followed byinjection of the fibronectin-based scaffold protein in solution andregeneration of the Protein A surface by glycine pH 2.0. Sensorgramswere obtained at each concentration and were evaluated using a programBiaevaluation, BIA Evaluation 2.0 (BIAcore), to determine the rateconstants k_(a) (k_(on)) and k_(d) (k_(off)) The dissociation constant,K_(D) was calculated from the ratio of rate constants k_(off)/k_(on).Typically, a concentration series (2 uM to 0 uM) of purifiedfibronectin-based scaffold protein was evaluated for binding to proteinA captured human IGF-1R-Fc fusion protein.

For experiments determining binding to human insulin receptor,recombinant human insulin receptor (IR) and recombinant human VEGF-R2-Fcwere directly coupled to a CM5 Biasensor chip by amine group linkagefollowing standard procedures recommended by Biacore (Uppsala, Sweden).In brief, 60 ug/mL of IR diluted in acetate 4.5 was coupled/immobilizedto a level of 8300 RU and 11.9 ug/mL of VEGF-R2-Fc diluted in acetate5.0 was immobilized to a level of 9700 RU on flow cells 2 and 3. A blankreference surface was prepared on FC1. Specific binding to either IR orVEGF-R2-Fc was calculated by subtracting the binding observed to theblank reference flow cell 1. Fibronectin-based scaffold proteins werediluted to 10 uM in HBS-EP (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05%Surfactant P20) and injected at 20 uL/min for 3 minutes over the flowcells at 25° C. and dissociation was observed over 10 mins.

Cell-Based Receptor Blocking Assay

The human breast adenocarcinoma MCF-7 (ATCC, Manassas, Va.) was platedin 24 well plates at a concentration of 50,000 cells per well in RPMI1640 (Invitrogen, Carlsbad, Calif.) containing 10% fetal bovine serum(Hyclone, Logan, Utah). The following day, cells were washed in bindingbuffer consisting of RPMI 1640 containing 0.1% BSA (Sigma, St. Louis,Mo.), and then pre-incubated for 30 minutes on ice in 200 μL bindingbuffer containing IGF-IR competitor. After the pre-incubation period, 40pM [¹²⁵I]-IGF-I (Perkin Elmer, Wellesley, Mass.), equivalent toapproximately 60000 counts per minute, was added to each well andallowed to incubate for an additional three hours on ice with gentleagitation. The wells were then washed with ice cold PBS containing 0.1%BSA. Cells were lysed with 500 μL buffer consisting of 0.1% SDS+0.5 NNaOH. Radioactivity of the lysates was measured using a Wallac 1470Gamma Counter (Perkin Elmer, Wellesley, Mass.), and the data wereanalyzed using SigmaPlot (Systat Software, Point Richmond, Calif.).

pIGFR Assay

Fibronectin-based scaffold proteins fused to Fc were evaluated for theirability to inhibit IGF-1R phosphorylation in Rh41 human rhabdomyosarcomacells. A Western Blot was employed to assess the ability of the Imonomer to inhibit IGF-1R phosphorylation in Rh41 human rhabdomyosarcomacells. Cells were stimulated with IGF-I, IGF-II, insulin ligands (50ng/ml), or no stimulation (NS) and then treated with variousconcentrations of the I monomer. Membranes were probed withphospho-specific antibodies.

Cellular Proliferation in Rh41 (Human Rhabdomyosarcoma and H929 HumanMultiple Myeloma)

Proliferation was evaluated by incorporation of [³H]-thymidine into DNAafter a 72 hour exposure to reagents. Rh41 cells were plated at adensity of 3500 cells/well in 96-well microtiter plates and 24 hourslater they were exposed to a range of I monomer concentrations. After 72hours incubation at 37° C., cells were pulsed with 4 μCi/ml [³H]thymidine (Amersham Pharmacia Biotech, UK) for 3 hours, trypsinized,harvested onto UniFilter-96, GF/B plates (PerkinElmer, Boston, Mass.)and scintillation was measured on a TopCount NXT (Packard, Conn.).Results are expressed as an IC50, which is the drug concentrationrequired to inhibit cell proliferation by 50% to that of untreatedcontrol cells Data represents the average of triplicate wells withstandard deviations shown.

Results of the characterization of the I monomer are shown below inTable 12.

TABLE 12 Properties of I monomers. IGFR Inhibition of BIAcoreNeutralizes Inhibition of RH41 KD IGF Binding pIGFR ProliferationMonomer IC50 IC50 IC50 IC50 I1 0.11 nM 8 nM 0.2 nM 28 nM

Example 20 Additional Characteristics of Monospecific and BispecificEGFR and IGF-IR ¹⁰Fn3-Based Binders

¹⁰Fn3-based binders that bound either EGFR or IGF-IR were identifiedusing the biochemical selection technique of mRNA display in which aprotein is covalently attached to its coding nucleic acid sequences.¹⁰Fn3-based proteins-mRNA fusion populations that bound either IGF-IR orEGFR when the receptors were presented at concentrations from 1 to 10 nMwere cloned into E. coli and expressed as ¹⁰Fn3-based proteins. A subsetof target binders that blocked EGFR or IGF-IR signaling and had suitablebiophysical properties were identified (Table 13). These initial cloneswere optimized for target binding affinity and cellular potency withadditional mRNA selection at increasingly lower target concentrationsand selection for lower dissociation rate constants. IC₅₀ valuesobtained during the selection procedures ranged from 9 to 304 nM,illustrating the opportunity for choosing molecules from a wide range ofpotency values for the construction of bi-specific ¹⁰Fn3-based binders.EGFR ¹⁰Fn3-based binders were tested by In-Cell Western screening assaysfor the blockade of phosphorylation of EGFR and ERK, a downstreamsignaling molecule of EGFR activation (methods similar to Example 1).Analogous studies were performed on optimized IGF-IR binders. OptimizedEGFR-binding clones (E3, E1, and E2) inhibited EGFR phosphorylation onY1068 and downstream phosphorylation of ERK on Y204 of p42/p44 in vitrowith IC₅₀ values ranging from 9 to 40 nM, potencies that were more than100-fold higher than the parental EGFR clone (Table 13, methods similarto Example 1).

I1 bound to IGF-IR with a K_(D) value of 0.11 nM and inhibitedIGF-1-stimulated IGF-IR phosphorylation with an IC₅₀ of 0.2 nM (Table13, methods similar to Example 6). The optimized IGF-IR and EGFRsingle-domain ¹⁰Fn3-based binders were >95% monomeric based on sizeexclusion chromatography, had melting temperatures >56° C. (Table 13,methods similar to Example 4), and exhibited minimal immunogenicpotential as predicted from EpiMatrix (<7 for five out of six loops), amatrix-based algorithm for T-cell epitope mapping (De Groot A S, Moise L(2007) Prediction of immunogenicity for therapeutic proteins: state ofthe art. Curr Opin Drug Discovery Devel 10:332-340). The ¹⁰Fn3-basedbinders E1, E2, and E3 were selected for further development, and hadEGFR binding constants in the range of 0.7 to 10 nM as determined fromBiacore assay (Table 13, methods similar to Example 5). EGFR-binding ofthese ¹⁰Fn3-based binders was competitive for EGF binding to EGFR (Table13) as measured by a displacement assay using Europium labeled EGF(methods similar to Example 10). Similarly, IGF-I binding to IGF-IR wasinhibited by I1 (Table 13, methods similar to Example 19).

Biophysical Characterization of Bi-Specific ¹⁰Fn3-based binders. T_(m)values of selected E/I ¹⁰Fn3-based binders ranged from 49-58° C. andtheir SEC profiles indicated the protein was >90% monomer (Table 14,methods similar to Example 4). Monospecific ¹⁰Fn3-based binders and E/I¹⁰Fn3-based binders showed comparable binding affinities, although T_(m)values decreased slightly when the single domain ¹⁰Fn3-based binderswere linked together (Tables 13 and 14). To increase serum half life forin vivo applications, E/I ¹⁰Fn3-based binders were PEGylated with a 40kDa branched PEG (methods similar to Example 3). PEGylation of E/I¹⁰Fn3-based binders resulted in a 10- to 20-fold reduction of bindingaffinity relative to the un-PEGylated constructs due to decreasedassociation rate constants but did not decrease T_(m). Furthermore,PEGylation did not markedly reduce inhibition of EGFR/IGF-IRphosphorylation in cells. The PEGylated E-I orientation (wherein theEGFR binder is at the N terminus, and IGF1R is at the C terminus)exhibited slightly lower IC₅₀ values for the inhibition of EGFR andIGF-IR phosphorylation by ELISA compared to the I-E orientation. Whileminor differences in the K_(D) values and biological activity were foundbetween PEGylated E-I orientation, vs the I-E orientation, there were noconsistent trends.

TABLE 13 Properties of Monospecific ¹⁰Fn3-based binders Relevant to theConstruction of E/I ¹⁰Fn3-based binders. A431 A431 EGFR IGF-IR pEGFRpERK H292 H292 Competition T_(m) SEC KD KD IC₅₀, IC₅₀, pEGFR pIGF-IREGFR/IGF-IR Name ° C. Monomer % nM nM nM nM IC₅₀, nM IC₅₀, nM IC₅₀, nME- 56 ND 42.5 2580 2370 1148 ± 21  ND   29 ± 12.73 parent E3 60 >80 3.4NA 15 ± 8 11 ± 7 22 ± 1 >7000 4.75 ± 1.77 E1 64 >95 9.92 NA 24 ± 7 13 ±3  9 ± 2 >3400 15.9 ± 2.97 E2 72 >95 0.7 NA  38 ± 15 40 ± 9 31 ± 1 >3400 9.4 ± 3.68 I- ND ND NA 1.8  ND ND ND ND 13** parent I1 61.5 >95 >62100.11 NA NA NA 0.2  8** ND, not done; NA, not applicable; SEC, sizeexclusion chromatography. *IC₅₀ values for EGFR and ERK phosphorylationlevles in A431 cells were determined by In-Cell Western assay (ICW).Phosphorylation levels of EGFR and IGF-IR in H292 cells were determinedby Enzyme-linked immunosorbent assay (ELISA). **Competition for IGF-IRbinding. Standard deviations are from 3-6 experiments.

TABLE 14 Properties of the E/I ¹⁰Fn3-based binders. IGF- H292 A431 IRH292 pIGF-IR pERK A431 EGF-EGFR T_(m) EGFR K_(D) pEGFR IC₅₀, IC₅₀, pEGFRCompetition Name ° C. K_(D) nM nM IC₅₀, nM nM nM IC₅₀, nM IC₅₀, nME3-GS10-I1 52 0.7 0.1 7 6 12 14  25 ± 6.5 E3-GS10- 52.5 10.4 0.74 10 640 42  80.5 ± 12.02 I1-PEG E1-GS10-I1 48 3.8 0.8 30 1 51 36 51 E1-GS10-49 57.9 2.4 123 4 295 297 396 ± 223 I1-PEG E2-GS10-I1 56 0.5 0.2 8 0.120 19  2.1 ± 0.57 E2-GS10- 57.5 10.1 1.17 32 0.3 78 77 56.5 ± 24.5I1-PEG I1-GS10- 60 3.6 0.46 47 0.8 118 97  128 ± 4.95 E2-PEG T_(m)measurements are from thermal scanning flurometry. K_(D) values are fromBiacore binding assays using recombinant EGFR or IGF-IR domains adsorbedon the chip. In-Cell Western assays (ICW) were conducted to determinethe ability of EI-Tandems to inhibit the phosphorylation of EGFR or ERKin A431 cells. Enzyme-linked immunosorbent assays (ELISA) were used todetermine the phosphorylation of EGFR or IGF-IR in H292 cells.

Example 21 Species Cross-Reactivity of E/I ¹⁰Fn3-Based Binders

Pegylated E/I ¹⁰Fn3-based binders were analyzed for their bindingaffinities to EGFR from mouse, rat and monkey using surface plasmonresonance (BIAcore) analysis (methods identical to Example 5). MouseEGFR was purchased from R&D systems (Minneapolis, Minn.), rat EGFR wasproduced in house, and monkey EGFR was purchased from KEMP (Frederick,Md.)

As shown in Table 15, all pegylated E/I ¹⁰Fn3-based binders bound tomouse, rat and monkey EGFR with low nanomolar affinities indicating thatall pegylated E/I binders are cross-reactive with human, mouse, rat andmonkey EGFR.

TABLE 15 KD (nM) KD (nM) KD (nM) Analyte (mouse EGFR) (rat EGFR) (monkeyEGFR) I1-GS10-E105 2.7 2.9 4.4 (pegylated) I1-GS10-E5 3.4 3.6 5.1(pegylated) I1-GS10-E4 5.5 3.7 3.9 (pegylated) E4-GS10-I1 6.9 5.6 5.7(pegylated) E2-GS10-I1 9.6 9.6 18.0 (pegylated) I1-GS10-E85 13.9 10.77.0 (pegylated)

Example 22 Characterization of Additional E/I ¹⁰Fn3-Based Binders

FIG. 43 summarizes various characteristics of additional E/I ¹⁰Fn3-basedbinders.

The pegylated E/I ¹⁰Fn3-based binders were tested to determineinhibition of EGF induced EGFR and ERK phosphorylation in A431, usingmethods as previously described in Example 1. Results demonstrated thatthe pegylated E/I ¹⁰Fn3-based binders inhibited EGF induced EGFRphosphorylation with IC50's ranging from 12 nM-297 nM andphosphorylation of ERK with IC50's ranging from 12 nM-295 nM (FIG. 43,columns a and b).

The ability of the pegylated E/I ¹⁰Fn3-based binders to inhibit IGFR andEGFR activity was also examined in H292 cells using methods previouslydescribed in Examples 6 and 7. Results indicated that the pegylated E/I¹⁰Fn3-based binders inhibited IGFR activity with IC50's ranging from 0.2nM-6 nM (FIG. 43, column d) and inhibited EGFR activity with IC50'sranging from 1.3 nM-123 nM (FIG. 43, columns c).

The pegylated E/I ¹⁰Fn3-based binders were tested to determine if theycould induce degradation of EGFR and IGFR in Difi cells as shown incolumns e and f of FIG. 43. Cells were treated with 1 uM of pegylatedE/I ¹⁰Fn3-based binders and harvested at time points starting at 7 hrsand ending at 120 hrs and levels of EGFR and IGF1R were determine byWestern blot analysis. The strength of degradation was scored as either(+) indicating the tandem degraded that receptor but the degradation wasnot sustained and receptor expression reappeared during the time courseor (++) which indicates the tandem degraded the receptor and sustainedthat degradation throughout the time course. Results (FIG. 43, column eand f) demonstrated that the pegylated E/I ¹⁰Fn3-based binders displayedvarious patterns of EGFR and IGF1R degradation; degradation of onlyIGFR, degradation of both EGFR and IGFR or no degradation of eitherreceptor. No tandem tested displayed the ability to degrade only EGFR.

The binding affinity of the pegylated E/I ¹⁰Fn3-based binders for EGFRand IGF1R was assessed by surface Plasmon resonance (BIAcore) analysisas previously described in Example 5. Results demonstrated that thepegylated E/I ¹⁰Fn3-based binders bound to EGFR with affinities rangingbetween 3.35 nM-57.9 nM and bound to IGF1R with affinities rangingbetween 0.37 nM-2.43 nM (FIG. 43, columns g and h).

The pegylated E/I ¹⁰Fn3-based binders were tested to determine theirpotency for blocking EGF binding to EGFR on the surface of A431 cellsusing methods previously described in Example 10. The pegylated E/I¹⁰Fn3-based binders blocked EGF binding to A431 cells with IC50'sranging from 19.5 nM to 238 nM (FIG. 43, column i).

The pegylated E/I ¹⁰Fn3-based binders were assessed for their ability toinhibit colony formation of H292 cells using methods described inExample 17. As shown in FIG. 43, column j, the pegylated E/I ¹⁰Fn3-basedbinders inhibited colony formation with IC50 values ranging from 1nM-560 nM and three of the four pegylated E/I ¹⁰Fn3-based binders testedwere 23-140 fold more potent than the anti-EGFR monoclonal antibodypanitumumab. The fourth pegylated E/I ¹⁰Fn3-based binders was 4 foldless potent than panitumuab. The pegylated I1 monomer was onlymarginally active in inhibiting colony formation in H292 with an IC50>15uM and this is expected since H292 cell growth is predominantly drivenby EGFR signaling and not IGF1R signaling.

The melting temperature was assessed for pegylated E/I ¹⁰Fn3-basedbinders by DSC (as previously described in Example 4) or thermal dyemelt methodology. For thermal dye melt assessment, the pegylated E/I¹⁰Fn3-based binders were diluted to 0.2 mg/mL in 50 mM NaAc buffer pH4.5. Each sample was spiked with 1 uL of the 200× Sypro Orange in DMSObuffer for a final concentration of 0.5% dye. Each sample was loadedinto the 96 well tray and coated with 5 uL of silicone oil. The tray wasspun down at 1,000 RPM and loaded onto the Bio-Rad CFX96 system and thefollowing method was selected: 25° C. for 10 minutes+Plate Read 25° C.to 95° C.@0.5° C. increments for 15 minutes+Plate Read. Data analysiswas performed for the inflection point with the CFX software. As shownin FIG. 43, column k, all pegylated E/I ¹⁰Fn3-based binders had similarTm measurements, ranging from 49-62.5 degrees celsius. Tm measurementsfor the pegylated E/I ¹⁰Fn3-based binders were independent ofconcentration and remained consistent at all concentrations tested. DSCanalysis of an exemplary binder, I1-GS10-E5 pegylated, measured with ascan range of 15-95° C. at 1 mg/ml protein concentration in PBS,resulted in a Tm measurement of 55.2° C. as shown in FIG. 20.

Size exclusion chromatography (SEC) was performed on the pegylated E/I¹⁰Fn3-based binders as previously described in Example 4. SEC analysisrevealed that all of the pegylated E/I ¹⁰Fn3-based binders were >95%monomeric as shown in FIG. 43 (column 1 of Table).

Example 23 Biochemical and Biophysical Properties of E/I ¹⁰Fn3-BasedBinder I1-GS10-E5 Pegylated with Selected Amino Acid Changes

I1-GS10-E5 pegylated was constructed without the 6HIS tag (SEQ ID NO:487) and also with various alterations to the linker region. Inaddition, a global change was made to all the constructs wherein theC-terminal tail of the first monomer had a single point change of theaspartic acid to glutamic acid (D to an E). Several clones were madewith selected serine residues mutated to cysteines (S to C) to providefor alternate PEGylation sites. The effect of these changes onbiochemical and biophysical properties of the molecule were compared andare summarized in Table 16. Methods for measuring inhibition of pEGFRare described in Example 7, pIGFR in Example 6, pERK in Example 1, Tm inExample 4, EGFR and IGFR KD in Example 5. Detailed analysis of thebinding kinetics were also carried out on these clones and are presentedin Tables 17 and 18 (using methods similar to those described in Example5).

TABLE 16 pEGFR pIGFR pERK EGFR IGFR IC50 IC50 IC50 Tm KD KD SEC % CLONENAME (nM) (nM) (nM) (° C.) (nM) (nM) mono I1-GS10-E5 pegylated 28 2.2 1256 2.7 0.25 96 I1-GS10-E5 30 1.2 11 56.8 Sticky⁽⁹⁾ 0.23 94.1pegylated⁽¹⁾ I1-GSGCGS8-E5⁽³⁾ 19.8 1.4 8 54.8 4 0.29 95.2I1-GS10-E5-GSGC⁽⁴⁾ 28.7 1.2 19 55 1.4 0.25 92.7 I1 (S62C)-GS10-E5⁽⁵⁾ 211.9 10 55.5 8.7 0.7 97.45 I1-GS10-E5 (S62C)⁽⁶⁾ 68.4 2.2 30 56 1.7 0.2696.12 I1 (S91C)-GS10-E5⁽⁷⁾ 22.7 6.2 15 52 17 7.16 95.98I1-GS10-E5(S91C)⁽⁸⁾ 22.6 2.1 29 50.5 17.9 0.28 93.39 ⁽¹⁾No His Tag wasused for this construct. ⁽²⁾a global change was made to all thealternative constructs of I1-GS10-E5 pegylated, wherein the C-terminaltail of the first monomer had a single point change of aspartic acid toglutamic acid (D to an E). ⁽³⁾The I1 mononer linked with GSGC (SEQ IDNO: 489) plus GS8 (SEQ ID NO: 494), to E5. ⁽⁴⁾I1 linked with GS10 to E5with GSGC (SEQ ID NO: 489) at the tail of E5. ⁽⁵⁾I1 linked with GS10 toE5, wherein the I1 has a single point change of serine to cysteine atposition 62. ⁽⁶⁾I1 linked with GS10 to E5, wherein the E5 has a singlepoint change of serine to cysteine at position 62. ⁽⁷⁾I1 linked withGS10 to E5, wherein the I1 has a single point change of serine tocysteine at position 91. ⁽⁸⁾I1 linked with GS10 to E5, wherein the E5has a single point change of serine to cysteine at position 91. ⁽⁹⁾Thisconstruct demonstrated non-specific binding to the flow cell so anaccurate determination of affinity was not possible in this experiment.

TABLE 17 Biacore binding of altered I1-GS10-E5 Pegylated clones toEGFR645-Fc. Description ka (1/Ms) kd (1/s) Kd (nm) Δka (fold) Δkd (fold)ΔKd (fold) I1-GS10-E5 Pegylated 2.93 ± 0.67E+04 7.24 ± 3.14E−0 5 2.69 ±1.53 — — — I1-GS10-E5 pegylated 2.27E+04 1.49E−04 6.6 0.8 0.5 0.4I1-GS10-E5 pegylated⁽¹⁾ Non-specific binding to reference cell surfaceat higher analyte concentrations (600 nM, 200 nM prohibited kineticvalue determination) ALTERNATIVE CLONES⁽²⁾ I1-GSGCGS8-E5⁽³⁾ 2.94E+041.18E−04 4.0 1.0 0.6 0.7 I1-GS10-E5-GSGC⁽⁴⁾ 3.34E+04 4.52E−05 1.4 1.11.6 2.0 I1(S62C)-GS10-E5⁽⁵⁾ 2.28E+04 1.99E−04 8.7 0.8 0.4 0.3I1-GS10-E5(S62C)⁽⁶⁾ 1.78E+04 3.04E−05 1.7 0.6 2.4 1.6I1(S91C)-GS10-E5⁽⁷⁾ 1.96E+04 3.34E−04 17.0 0.7 0.2 0.2I1-GS10-E5(S91C)⁽⁸⁾ 1.08E+04 1.93E−04 17.9 0.4 0.4 0.2 ⁽¹⁾No His Tag wasused for this construct. ⁽²⁾a global change was made to all thealternative constructs of I1-GS10-E5 pegylated, wherein the C-terminaltail of the first monomer had a single point change of aspartic acid toglutamic acid (D to an E). ⁽³⁾The I1 mononer linked with GSGC (SEQ IDNO: 489) plus GS8 (SEQ ID NO: 494), to E5. ⁽⁴⁾I1 linked with GS10 to E5with GSGC (SEQ ID NO: 489) at the tail of E5. ⁽⁵⁾I1 linked with GS10 toE5, wherein the I1 has a single point change of serine to cysteine atposition 62. ⁽⁶⁾I1 linked with GS10 to E5, wherein the E5 has a singlepoint change of serine to cysteine at position 62. ⁽⁷⁾I1 linked withGS10 to E5, wherein the I1 has a single point change of serine tocysteine at position 91. ⁽⁸⁾I1 linked with GS10 to E5, wherein the E5has a single point change of serine to cysteine at position 91.

TABLE 18 Biacore binding of altered I1-GS10-E5 Pegylated clones toIGF1R-Fc. Description ka (1/Ms) kd (1/s) Kd (nm) Δka (fold) Δkd (fold)ΔKd (fold) I1-GS10-E5 pegylated 1.04 ± 0.04E+06 2.62 ± 0.21E−04 0.25 ±0.01 — — — I1-GS10-E5 pegylated 1.10E+06 2.78E−04 0.25 1.1 0.9 1.0I1-GS10-E5 pegylated⁽¹⁾ 1.28E+06, 2.88E−04, 0.22, 0.23 1.2 0.9 1.11.22E+06 2.76E−04 ALTERNATIVE CLONES⁽²⁾ I1-GSGCGS8-E5⁽³⁾ 8.52E+052.45E−04 0.29 0.8 1.1 0.9 I1-GS10-E5-GSGC⁽⁴⁾ 1.07E+06 2.65E−04 0.25 1.01.0 1.0 I1(S62C)-GS10-E5⁽⁵⁾ 3.34E+05 2.34E−04 0.70 0.3 1.1 0.4I1-GS10-E5(S62C)⁽⁶⁾ 1.07E+06 2.79E−04 0.26 1.0 0.9 1.0I1(S91C)-GS10-E5(7) 8.22E+04 5.89E−04 7.16 0.1 0.4  0.04I1-GS10-E5(S91C)⁽⁸⁾ 9.86E+05 2.81E−04 0.28 0.9 0.9 0.9 ⁽¹⁾No His Tag wasused for this construct. ⁽²⁾a global change was made to all thealternative constructsof I1-GS10-E5 pegylated, wherein the C-terminaltail of the first monomer had a single point change of aspartic acid toglutamic acid (D to an E). ⁽³⁾The I1 mononer linked with GSGC (SEQ IDNO: 489) plus GS8 (SEQ ID NO: 494), to E5. ⁽⁴⁾I1 linked with GS10 to E5with GSGC (SEQ ID NO: 489) at the tail of E5. ⁽⁵⁾I1 linked with GS10 toE5, wherein the I1 has a single point change of serine to cysteine atposition 62. ⁽⁶⁾I1 linked with GS10 to E5, wherein the E5 has a singlepoint change of serine to cysteine at position 62. ⁽⁷⁾I1 linked withGS10 to E5, wherein the I1 has a single point change of serine tocysteine at position 91. ⁽⁸⁾I1 linked with GS10 to E5, wherein the E5has a single point change of serine to cysteine at position 91.

Example 24 Inhibition of Shared Downstream Signaling Pathways of EGFRand IGFR

Inhibition of downstream signaling pathways were analyzed with a pAKTELISA identical to those previously described in Example 8. Results ofthis study demonstrate that I1-GS10-E5 pegylated is more potent than I1pegylated alone at blocking IGF1-stimulated AKT activation in H292cells. E5 pegylated, the EGFR monospecific binder alone did notefficiently prevent activation of AKT by IGF1 stimulation (FIG. 21).

Example 25 Inhibition of Cell Proliferation by ¹⁰Fn3-Based Binders andComparator Antibody

H292 and RH41 cell proliferation experiments were conducted as describedin Example 9. The EGFR monospecific ¹⁰Fn3-based binder E5-pegylatedinhibited proliferation of H292 cells with an IC50 value of 0.016 μM.The IGFR monospecific ¹⁰Fn3-based binder I1-pegylated had an IC50 valueof >8.4 μM while the E/I ¹⁰Fn3-based binder I1-GS10-E5 pegylated wasslightly more potent with an IC50 value of 0.006 μM (FIG. 22). The H292cell line is of lung cancer origin and sensitive to inhibition of IGFRand EGFR ((Akashi Y, et al. (2008) Enhancement of the antitumor activityof ionising radiation by nimotuzumab, a humanised monoclonal antibody tothe epidermal growth factor receptor, in non-small cell lung cancer celllines of differing epidermal growth factor receptor status. Br. J.Cancer 98:749-755; and Buck E, et al. (2008) Feedback mechanisms promotecooperativity for small molecule inhibitors of epidermal andinsulin-like growth factor receptors. Cancer Res. 68:8322-8332.)) Incontrast, only the I1-GS10-E5 pegylated binder and the I1-pegylatedbinder inhibited the proliferation of RH41 cells (IC50 values were0.0002 and 0.0004 μM, respectively, FIG. 23). This was expected, sinceRH41 is a pediatric rhabdomyosarcoma cell line that is known to bedriven predominantly by IGFR signaling ((Huang F, et al. (2009). Themechanisms of differential sensitivity to an insulin-like growthfactor-1 receptor inhibitor (BMS-536924) and rationale for combiningwith EGFR/HER2 inhibitors. Cancer Res. 69:161-170)) and thus notsensitive to EGFR blockade.

Example 26 Inhibition of Receptor Activation and Downstream Signaling InVitro by Pegylated and Non-Pegylated ¹⁰Fn3-Based Binders

In order to understand the dynamics of EGFR/IGFR signaling and itsinhibition by I1-GS10-E5 pegylated, DiFi, H292 or BxPC3 cells wereserum-starved, exposed to 1 μM or 0.1 μM E5 pegylated, I1 pegylated, orI1-GS10-E5 pegylated, or vehicle control for 2 hours, then stimulatedwith either EGF, IGF-I, or EGF+IGF-I for 10 min.

Cells were cultured in vitro, serum starved overnight and then exposedto ¹⁰Fn3-based binders for 2 hours prior to stimulation with 100 ng/mlof EGF or IGF. Cell lysates were prepared in lysis buffer (1% TritonX-100, 5% glycerol, 0.15 M NaCl, 20 mM Tris-HCl pH 7.6, CompleteProtease Inhibitor Cocktail Tablets [Roche, Indianapolis, Ind.] andPhosphatase Inhibitor Cocktail 2 [Sigma-Aldrich Corp.]). Lysates (30 μg)were resolved by SDS-PAGE, transferred to membranes, and immunoblottedwith antibodies to phospho-EGFR and total EGFR (Santa CruzBiotechnology, Carlsbad, Calif.), phospho-AKT (Ser 473), phospho-p44/42MAPK (Thr202/Tyr204) (Cell Signaling Technology, Beverly, Mass.), ortotal actin (Chemicon International, Temecula, Calif.) in OdysseyBlocking Buffer with 0.1% Tween 20 (LI-COR Biosciences, Lincoln, Nebr.).Membranes were incubated with the appropriate secondary antibodies.Protein visualization was performed using a LI-COR Biosciences Odysseyinfrared imaging system.

As shown in FIG. 24, the basal levels of phosphorylated EGFR, IGF-IR,and AKT were nearly undetectable after serum deprivation. In DiFi cells,neither I1-GS10-E5 pegylated or E5 pegylated (monospecific EGFR binder)are able to completely suppress EGF-stimulated EGFR phosphorylation. InH292 and BxPC3 cells there is strong inhibition of EGFR phosphorylationby both I1-GS10-E5 pegylated and E5 pegylated. In DiFi and BxPC3 cells,I1-GS10-E5 pegylated blocks IGF-stimulated IGFR phosphorylation morethan I1 pegylated (monospeciific IGFR binder) by itself. In H292 cells,IGF-stimulation cross activates the EGFR only when EGFR is blocked.I1-GS10-E5 pegylated inhibited EGF-stimulated pAKT in DiFi; increasedpAKT in EGF-stimulated H292 and in BxPC3 EGF did not activate pAKT. InDiFi, H292 and BxPC3 cells I1-GS10-E5 pegylated inhibited IGF-stimulatedpIGFR more than the individual E5 pegylated and I1 pegylated bythemselves. I1-GS10-E5 pegylated had very little if any effect onEGF-stimulated pERK in DiFi, H292 or BxPC3. IGF-stimulation did notinduce pERK in any cell line examined.

In another experiment with unPEGylated_(—) ¹⁰Fn3-based binders, H292cells were serum-starved, exposed to 1 μM unPEGylated monospecific EGFRbinder E2, IGFR binder I1, or E2-GS10-I1, or vehicle control for 1 hour,then stimulated with either EGF, IGF-I, or EGF+IGF-I for 10 min. Thebasal levels of phosphorylated EGFR, IGFR, and AKT were nearlyundetectable after serum deprivation (FIG. 25). Stimulation with EGFinduced EGFR phosphorylation, but did not transactivate IGFR. EGFRphosphorylation was blocked by the E2, and E2-GS10-I1, but not ILSimilarly, stimulation with IGF-I induced strong phosphorylation of IGFRthat was blocked by I1 and E2-GS10-I1, but not by E2. EGF stimulationonly slightly increased AKT phosphorylation, but IGF-I or EGF+IGF-Istrongly induced phosphorylation of AKT that was suppressed to basallevels by both I1 and E2-GS10-I1. The combination of IGF-I and EGFinduced AKT phosphorylation more than either growth factor alone. E2partially reduced pAKT induced by the combination of EGF and IGF-I.However, I1 showed the most dramatic reduction in pAKT, suggesting thatstimulation with EGF+IGF-I led to strong AKT phosphorylation through theIGFR pathway. Surprisingly, blockade of the EGFR pathway by E2 followedby stimulation with EGF ligand actually increased the phosphorylation ofAKT, perhaps as a result of EGFR-independent activation of AKT ((DobashiY, et al. (2009) EGFR-dependent and independent activation of Akt/mTORcascade in bone and soft tissue tumors. Mod Pathol (Epub Ahead ofPrint)). These results illustrate the complex cross-talk between theEGFR and IGFR pathways and feed-back mechanisms.

Example 27 Competition Binding Studies with E/I ¹⁰Fn3-Based Binders

For Biacore competition experiments, EGFR-Fc (3 μg/mL in Na-acetate pH5.0) was immobilized on the Biacore CM5 chip surface using standardEDC/NHS amide coupling chemistry to a surface density of 300 RU. EGFRantibodies were obtained as a marketed drug and competition betweenmonospecific EGFR binder E2 and antibodies for binding to EGFR-Fc wasassessed by binding 450 nM E2 (30 μL/min, 200s contact time),immediately followed by 450 nM E2 alone, or a mixture of 450 nM E2 plus450 nM cetuximab, panitumumab, or nimotuzumab (30 μL/min, 200 seccontact time). The surface was successfully regenerated between cyclesusing two 10 sec pulses of 50 mM NaOH at a flow rate of 30 μL/min.Initial injection of E2 shows binding to EGFR on the surface of thechip. A second injection of E2 mixed with an equal amount of cetuximab,panitumumab, or nimotuzumab shows no competition for binding ofantibodies to EGFR by E2 (FIG. 26A).

Surface plasmon resonance (BIAcore) analysis was utilized to demonstratesimultaneous engagement of captured EGFR-Fc and solution phase IGF1R byE/I ¹⁰Fn3-based binders. Recombinant human EGFR-Fc (aa 1-645 of theextracellular domain of human EGFR fused to human Fc) was purchased fromR&D systems (Minneapolis, Minn.). Recombinant IGF1R (aa 1-932 of humanIGF1R propeptide, proteolytically cleaved and disulfide linked) waspurchased from R&D systems (Minneapolis, Minn.). To demonstratesimultaneous engagement, anti-human IgG was immobilized on flow cells 1and 2 of a CM5 chip following the manufacturer's recommendations (GEHealthcare, Piscataway, N.J.). EGFR-Fc (50 nM) was captured on flow cell2 at 10 uL/min for 2 minutes. Binding of E/I ¹⁰Fn3-based binders toEGFR-Fc was achieved by injecting ¹⁰Fn3-based protein samples (100 nM)over both flow cells at 10 uL/min for 2 minutes. Simultaneous engagementof EGFR-Fc and IGF1R was probed by subsequently injecting IGF1R (0.100nM) over both flow cells at 30 uL/min for 2 minutes. Dissociation of thecomplex was monitored for 300 seconds. Two 30 second injections of 3 MMgCl₂ were used for regeneration of the bound complex from theanti-human IgG surface. Biacore T100 Evaluation Software, Version 2.0.1(GE healthcare/Biacore) was utilized to overlay sensograms and removeairspikes. As shown in FIG. 26B, both domains of the E/I ¹⁰Fn3-basedbinder are functional and able to bind to EGFR-Fc and IGF1Rsimultaneously.

Binding specificity of E2-GS10-I1 pegylated to HER family receptors wasassessed by Biacore as described in Example 5. HER-2-Fc, HER-3-Fc andHER-4-Fc (R&D Systems) was captured on the surface of the CM5 chip withanti-human IgG. E2-GS10-I1 pegylated did not show any discerniblebinding to other HER family members under conditions where robustbinding was seen for EGFR-Fc (HER-1) (Table 19).

TABLE 19 Binding affinity of E2-GS10-I1 pegylated to extracellulardomains of HER family of receptors. HER- HER- HER- EGFR- 2-Fc 3-Fc 4-FcFc K_(D), K_(D), K_(D), K_(D), Name nM* nM nM nM E2-GS10-I1pegylated10.1 >1000 >1000 >1000

Example 28 Measurement of Plasma Biomarkers

Levels of soluble biomarkers TGFα and mIGF1 were measured in mouseplasma at the end of xenograft studies or in non tumor bearing mice atvarious times following treatment. Blood was obtained by terminalcardiac puncture into tubes containing EDTA as an anticoagulant. Plasmawas prepared by centrifuging blood at 1300×g for 10 minutes at 4 degreesC. and removing the clarified supernatant to a separate tube. TGFαlevels were measured in 0.1 ml of plasma, mIGF1 levels were measured in0.02 ml plasma with an ELISA assay as recommended by manufacturer (R&DSystems, Minneapolis, Minn.). Plasma levels of TGFα were increased inmice treated with I1-GS10-E5 pegylated or the monospecific EGFR binderE5 pegylated but not cetuximab (FIG. 27A-C). The TGFα could be secretedfrom the human tumor or may represent endogenous mouse TGFα. Due to thehigh homology between human and mouse TGFα (93% amino acid identity) theELISA may cross react with mouse TGFα. Furthermore, human TGFα secretedby the implanted tumor can bind to the mouse EGFR. Because I1-GS10-E5pegylated and E5 pegylated can bind both human and mouse EGFR, all hostand tumor EGFR binding sites are blocked by these ¹⁰Fn3-based binderswhile cetuximab does not bind mouse EGFR. To determine if these¹⁰Fn3-based binders cause increases in endogenouse mouse TGFα and if theELISA cross reacts with mouse TGFα, non-tumor bearing nude mice weredosed with I1-GS10-E5 pegylated at 100 mg/kg and plasma samples weretaken at 4, 24, 48, 72 hours post dose. Increases in mouse TGFα were infact observed that persisted out past 72 hours (FIG. 28A). Plasmasamples from non-tumored mice were also tested for mIGF1 with a mousespecific ELISA and increases in this ligand were also observed (FIG.28B).

Example 29 Results of In Vivo Human Tumor Xenograft Studies for VariousE/I ¹⁰Fn3-Based Binders

Several E/I ¹⁰Fn3-based binders were evaluated in a head-to-head H292NSCLC study (methods described in Example 12) at a lower dose thanpreviously used so that differences in relative activity could beascertained. Efficacy of the E/I ¹⁰Fn3-based binders E2-GS10-I1pegylated, E4-GS10-I1 pegylated, I1-GS10-E5 pegylated, I1-GS10-E85pegylated, I1-GS10-E4 pegylated, I1-GS10-E105 pegylated at a single doseof 0.625 mg/mouse and panitumumab at two doses (1 mg/mouse and 0.1mg/mouse) were compared.

Both doses of panitumumab and all E/I ¹⁰Fn3-based binders evaluated inthis study were active by a tumor growth inhibition (TGI) endpoint.During the dosing phase, E4-GS10-I1 pegylated, I1-GS10-E5 pegylated,I1-GS10-E4 pegylated and panitumumab all caused tumor regression (Table20, TGI values greater than 100%) while E2-GS10-I1 pegylated,I1-GS10-E85 pegylated and I1-GS10-E105 pegylated caused tumor growthinhibition (Table 20, TGI values up to 100%). Differences in activitywere statistically significant when compared to the control group. Alltreatments were well tolerated with no treatment related deaths orexcessive weight loss over the course of the study. Comparison of theefficacy of the E/I ¹⁰Fn3-based binders and panitumumab are presented inTable 20 below and in FIG. 29. In FIG. 29A, measurements out to day 43shows the pattern of regrowth of the tumors after dosing ceased. FIG.29B shows measurements out to day 27 and the y-axis is expanded toillustrate the relative differences in activity among the treatmentgroups.

TABLE 20 In vivo antitumor activity in the H292 NSCLC study Schedule,Dose AVE weight p value for Outcome Group Compound Route (mg/kg) change(g) % TGI % TGI by % TGI 1 Control — — 3.36 — 1.0 — (untreated) 2panitumumab q3dx5; 6 ip^(a)    1 mg/mse 5.19 107 0.0023 A 3 panitumumabq3dx5; 6 ip^(a)  0.1 mg/mse 5.9 105 0.0029 A 4 E2-GS10-I1 TIWX3; 6ip^(a) 0.625 mg/mse −1.4 93 0.0067 A pegylated 5 E4-GS10-I1 TIWX3; 6ip^(a) 0.625 mg/mse −0.23 105 0.0023 A pegylated 6 I1-GS10-E5 TIWX3; 6ip^(a) 0.625 mg/mse −2.92 103 0.0033 A pegylated 7 I1-GS10-E85 TIWX3; 6ip^(a) 0.625 mg/mse 1.08 86 0.0114 A pegylated 8 I1-GS10-E4 TIWX3; 6ip^(a) 0.625 mg/mse −1.21 103 0.0034 A pegylated 9 I1-GS10-E105 TIWX3; 6ip^(a) 0.625 mg/mse −1.54 95 0.0035 A pegylated ^(a)Vehicle wasphosphate buffered saline. Abbreviations used are as follows: ip,intraperitoneal route; % TGI, relative % tumor growth inhibitioncalculated as % TGI = [(C_(t) − T_(t))/(C_(t) − C₀)] × 100 where C_(t) =median tumor weight of control mice at time t in days after tumorimplant, T_(t) = median tumor weight of treated mice at time t, C₀ =median tumor weight of control mice at time 0. % TGI value wascalculated at two points as the average inhibition of Day 20, Day 24 andDay 27. Outcome, a treatment regimen was considered active if itproduced a statistically significant % TGI value of >50%; q3dx5; 6,compound was administered every three days for six doses starting on thesixth day after tumor implant; 6 on/1 off; 6, compound was administeredonce a day for 6 days then no treatment for 1 day and this regimenstarted on the sixth day after tumor implant. p values were calculatedon Day 20 relative to the control group in a two tailed paired analysiswith 8 measurements per group.

Further in vivo studies were carried out with selected E/I ¹⁰Fn3-basedbinders below, in various xenograft models using the methods describedin Example 12. A description of the various xenograft models is asfollows: H292 is a non-small cell lung carcinoma (NSCLC) and isdescribed in more detail Example 12; MCF7r breast carcinoma is describedin Example 14; and GEO colon carcinoma is described in Example 15. TheDiFi human colon carcinoma expresses high levels of activated EGFR andalso expresses IGFR; RH41 is a pediatric rhabdomyosarcoma cell line thatis known to be driven predominantly by IGFR signaling (Huang F, et al.((2009)) The mechanisms of differential sensitivity to an insulin-likegrowth factor-1 receptor inhibitor (BMS-536924) and rationale forcombining with EGFR/HER2 inhibitors. Cancer Res. 69:161-170) and thus isnot sensitive to EGFR blockade; Cal27 is a human head and neck carcinomaexpressing high levels of EGFR and moderate levels of IGFR; BxPC3 is ahuman pancreatic carcinoma; and H441 is a NSCLC.

Comparison of the efficacy of selected E/I ¹⁰Fn3-based binders arepresented in Table 21. In these efficacy studies, all of the E/I¹⁰Fn3-based binders showed equivalent activity to panitumumab and alltreatments were able to regress H292 tumors below their starting size asindicated by % TGI values over 100%. In the DiFi study, panitumumabregressed tumors at the 1 mg/mouse dose and was active at the 0.1mg/mouse dose while all of the E/I ¹⁰Fn3-based binders were inactivealthough the I1-GS10-E5 pegylated showed some inhibition of tumor growth(TGI=43.8%). In the RH41 study, panitumumab was not active at eitherdose, the E2-pegylated construct was not active while the E/I¹⁰Fn3-based binders and the I1-pegylated construct were all active. FIG.32 shows antitumor efficacy in the RH41 model for a representativeconstruct E2-GS10-I1 pegylated (data also shown in Table 22). In theCal27 study panitumumab regressed tumors at the 1 mg/mouse dose and wasactive at the 0.1 mg/mouse dose but among the E/I ¹⁰Fn3-based bindersonly the I1-GS10-E5 pegylated E/I ¹⁰Fn3-construct was active.

Results of human tumor xenograft studies with I1-GS10-E5 pegylated andindividual 11 and E5 components designed to assess synergy are presentedin Table 22. These combination (synergy) studies were structured suchthat the individual pieces of the E/I ¹⁰Fn3-based binders (ie., IGFR andEGFR monospecific pegylated versions) were included so antitumor effectsbeyond the contribution of isolated ends could be discerned. In theMCF7r study, I1 pegylated was not active while the E5 pegylated, (E5pegylated+I1 pegylated) and the I1-GS10-E5 pegylated clones were allactive and exhibited similar activity meaning that all of the antitumoractivity likely comes from inhibition of EGFR and blocking the IGFRpathway did not provide any enhancement. Cetuximab regressed tumors atthe 1 mg/mouse dose and was not active at the 0.1 mg/mouse dose.BMS-754807 was also not active showing that blocking the IGFR pathwaywith a small molecule inhibitor did not result in efficacy in thismodel.

In the BxPC3 study, I1 pegylated was not active while the E5 pegylatedand (E5 pegylated+I1 pegylated) clones were active (TGI=61.2% and 68.8%,respectively). The I1-GS10-E5 pegylated clone was more active (TGI=78%)than the individual pieces it is made from and the difference wasstatistically significant by a two tailed paired t-test showing that ithas synergistic activity in this model. Cetuximab was active at alldoses studied but adding in IGFR inhibition by combining it with theI1-pegylated did not result in synergy.

In the GEO study, I1 pegylated was not active while the E5 pegylated and(E5 pegylated+I1 pegylated) and I1-GS10-E5 pegylated clones were active(TGI=83.5%, 92.1 and 92.1%, respectively). While there may have beensome enhancement provided by combining EGFR and IGFR inhibition togetherin this model, the difference was not significantly better than the E5pegylated by itself. Cetuximab was active at both doses studied butadding in IGFR inhibition by combining it with the I1-pegylated did notresult in synergy.

In the H441 study, I1 pegylated and E5 pegylated were not active ontheir own but (E5 pegylated+I1 pegylated) was active (TGI=54.5%). TheI1-GS10-E5 pegylated clone was more active (TGI=69.2%) than theindividual pieces it is made from but the differences were notstatistically significant showing that it provides enhanced activity butnot synergy in this model. Cetuximab was active at the 1 mg/mouse doseand was not active at the 0.1 mg/mouse dose. Adding in IGFR inhibitionby combining it with the I1-pegylated did not result in any enhancementin this model.

TABLE 21 In vivo results of selected E/I ¹⁰Fn3-based binders Dose AVEweight p value for Outcome Group Compound Schedule (mg/kg)^(a) change(g) % TGI % TGI by % TGI In vivo antitumor activity in the H292 study 1Control (untreated) — — 5.7 — 1.0 — 2 panitumumab Q3dx5; 6 ip^(a) 1mg/mse 5.0 104 0.0006 A 3 panitumumab Q3dx5; 6 ip^(a) 0.1 mg/mse   1.5102 0.0005 A 4 E4-GS10-I1 pegylated TIWX3; 6 2 mg/mse −4.86 105 0.0004 A5 I1-GS10-E5 pegylated TIWX3; 6 2 mg/mse −10.0 102 0.0006 A 6 I1-GS10-E4pegylated TIWX3; 6 2 mg/mse −1.41 105 0.0005 A In vivo antitumoractivity in the DiFi study 1 Control (untreated) — — −0.5 — 1.0 — 2panitumumab Q3dx5; 6 ip^(a) 1 mg/mse 5.3 109.7 0.006 A 3 panitumumabQ3dx5; 6 ip^(a) 0.1 mg/mse   2.6 99.9 0.005 A 4 E4-GS10-I1 pegylatedTIWX3; 6 3 mg/mse −10.8 −1.1 0.815 I 5 I1-GS10-E5 pegylated TIWX3; 6 3mg/mse −16.4 43.8 0.310 I 6 I1-GS10-E4 pegylated TIWX3; 6 3 mg/mse −8.51.4 0.977 I In vivo antitumor activity in the RH41study 1 Control(untreated) — — 7.2 — 1.0 — 2 panitumumab q3dx5; 6 ip^(a) 1 mg/mse 10.716.5 0.721 I 3 panitumumab q3dx5; 6 ip^(a) 0.1 mg/mse   8.6 38.4 0.563 I4 E4-GS10-I1 pegylated TIWX3; 6 2.5 mg/mse   −5.3 72.7 0.02 A 5I1-GS10-E5 pegylated TIWX3; 6 2.5 mg/mse   −7.8 68 0.019 A 6 I1-GS10-E4pegylated TIWX3; 6 2.5 mg/mse   −2.9 64.5 0.018 A 7 Control (untreated)— — 12.3 — 1.0 — 8 E2-GS10-I1 pegylated TIWX3; 18 2.5 mg/mse   −1.8 58.60.044 A 9 E2-pegylated TIWX3; 18 1.25 mg/mse   5.9 20.2 0.530 I 10 I1pegylated TIWX3; 18 1.25 mg/mse   7.1 58.6 0.025 A In vivo antitumoractivity in the Cal27 study 1 Control (untreated) — — 9.4 — 1.0 — 2Panitumumab q3dx5; 6 ip^(a) 1 mg/mse 6.1 109.8 0.0006 A 3 panitumumabq3dx5; 6 ip^(a) 0.1 mg/mse   5.8 72.9 0.003 A 4 E4-GS10-I1 pegylatedTIWX3; 6 2 mg/mse −1.2 −11.4 0.587 I 5 I1-GS10-E5 pegylated TIWX3; 6 2mg/mse −11.6 57.6 0.037 A 6 I1-GS10-E4 pegylated TIWX3; 6 2 mg/mse −2.2−9.2 0.177 I ^(a)Vehicle was phosphate buffered saline for alltreatments. Abbreviations used are as follows: ip, intraperitonealroute; po, oral route; % TGI, relative % tumor growth inhibitioncalculated as % TGI = [(Ct − Tt)/(Ct − C0)] × 100 where Ct = mediantumor weight of control mice at time t in days after tumor implant, Tt =median tumor weight of treated mice at time t, C0 = median tumor weightof control mice at time 0. % TGI value was calculated at two points asthe average inhibition on Day 19 and 23 for H292, Day 39 and 41 forDiFi, Day 34 and 37 for RH41 for groups 1-6 and Day 35, 36 and 39 forgroups 7-10, Day 18 and 20 for Cal27. Outcome, a treatment regimen wasconsidered active if it produced a statistically significant % TGI valueof >50%; q3dx5; 6, compound was administered every three days for sixdoses starting on the sixth day after tumor implant; TIWX3; 6, compoundwas administered three times a week for 3 weeks and this regimen startedon the sixth day after tumor implant. p values were calculated relativeto the control group in a two tailed paired analysis with 8 measurementsper group on Day 23 for H292, Day 39 for DiFi, Day 37 for RH41 forgroups 1-6 and Day 39 for groups 7-10 and Day 20 for Cal27.

TABLE 22 Summary of in vivo experiments with ¹⁰Fn3-based binders andcomparators Dose AVE weight p value for Outcome Group Compound Schedule(mg/kg)^(a) change (g) % TGI % TGI by % TGI In vivo antitumor activityin the MCF7r study 1 Control (untreated) — — 6.1 — 1.0 — 2 I1pegylated^(a) TIWX3; 7   50 mg/kg, ip 17.1 −40.8 0.195 I 3 E5pegylated^(a) TIWX3; 7   50 mg/kg, ip 5.1 75.8 0.007 A 4 E5pegylated^(a) + I1 pegylated^(a) TIWX3; 7   50 mg/kg, ip −3.0 81.8<0.0001 A 5 I1-GS10-E5 pegylated^(a) TIWX3; 7  100 mg/kg, ip −4.5 780.009 A 6 cetuximab^(a) Q3DX5; 7   1 mg/mse, ip 11.7 105.4 0.0009 A 7cetuximab^(a) Q3DX5; 7  0.1 mg/mse, ip 7.5 34.3 0.031 I 8 BMS-754807^(b)QDX14; 7   50 mg/kg, po −4.0 44.5 0.146 I In vivo antitumor activity inthe BxPC3 study 1 Control (untreated) — — 3.1 — 1.0 — 2 I1 pegylatedTIWX3; 9   50 mg/kg, ip 4.3 14.3 0.315 I 3 E5 pegylated TIWX3; 9   50mg/kg, ip −5.3 61.2 0.0003 A 4 E5 pegylated + I1 pegylated TIWX3; 9   50mg/kg, ip −4.9 68.8 0.0019 A 5 I1-GS10-E5 pegylated TIWX3; 9  100 mg/kg,ip −14.0 78.0 0.0002 A 6 cetuximab Q3DX5; 9   1 mg/mse, ip 5.2 62.60.0026 A 7 cetuximab Q3DX5; 9 0.25 mg/mse, ip 2.5 62.8 0.0005 A 8cetuximab + Q3DX5; 9   1 mg/mse, ip 3.6 62.1 0.0005 A I1 pegylatedTIWX3; 9   50 mg/kg, ip In vivo antitumor activity in the GEO study 1Control (untreated) — — 7.5 — 1.0 — 2 I1 pegylated TIWX3; 9   50 mg/kg,ip −7.2 26.8 0.594 I 3 E5 pegylated TIWX3; 9   50 mg/kg, ip 9.7 83.50.0028 A 4 E5 pegylated + I1 pegylated TIWX3; 9   50 mg/kg, ip 5.4 92.10.0005 A 5 I1-GS10-E5 pegylated TIWX3; 9  100 mg/kg, ip −7.3 92.1 0.0006A 6 cetuximab Q3DX5; 9   1 mg/mse, ip 7.7 91.8 0.0008 A 7 cetuximabQ3DX5; 9 0.25 mg/mse, ip 7.8 92.0 0.0007 A 8 cetuximab + Q3DX5; 9   1mg/mse, ip 7.1 91.3 0.0006 A I1 pegylated TIWX3; 9   50 mg/kg, ip Invivo antitumor activity in the H441 study 1 Control (untreated) — — 12.4— 1.0 — 2 I1 pegylated TIWX3; 9   50 mg/kg, ip 11.5 30.8 0.701 I 3 E5pegylated TIWX3; 9   50 mg/kg, ip −8.8 43.1 0.292 I 4 E5 pegyalted + I1pegylated TIWX3; 9   50 mg/kg, ip −0.8 54.5 0.011 A 5 I1-GS10-E5pegylated TIWX3; 9  100 mg/kg, ip −3.9 69.2 0.022 A 6 cetuximab Q3DX5; 9  1 mg/mse, ip 12.6 65.2 0.002 A 7 cetuximab Q3DX5; 9 0.25 mg/mse, ip13.7 43.9 0.110 I 8 cetuximab + Q3DX5; 9   1 mg/mse, ip 10.2 66.7 0.060I I1 pegylated TIWX3; 9   50 mg/kg, ip ^(a)Vehicle was phosphatebuffered saline for all treatments. Abbreviations used are as follows:ip, intraperitoneal route; po, oral route; % TGI, relative % tumorgrowth inhibition calculated as % TGI = [(Ct − Tt)/(Ct − C0)] × 100where Ct = median tumor weight of control mice at time t in days aftertumor implant, Tt = median tumor weight of treated mice at time t, C0 =median tumor weight of control mice at time 0. % TGI value wascalculated at two points as the average inhibition on Day 22 and 26 forMCF7r, Day 23 and 27 for BxPC3, Day 29 and 31 for GEO and Day 17 and forH441. Outcome, a treatment regimen was considered active if it produceda statistically significant % TGI value of >50%; q3dx5; 6, compound wasadministered every three days for six doses starting on the sixth dayafter tumor implant; TIWX3; 6, compound was administered three times aweek for 3 weeks and this regimen started on the sixth day after tumorimplant. p values were calculated relative to the control group in a twotailed paired analysis with 8 measurements per group on Day 26 forMCF7r, Day 27 for BxPC3, Day 29 for GEO and Day17 for H441.

Example 30 Pharmacokinetic Profile of Various E/I ¹⁰Fn3-Based Binders inMice

The pharmacokinetic profiles of the pegylated E/I ¹⁰Fn3-based binder,E2-GS10-I1, were assessed in mice via intraperitoneal injection. Threenude mice per dose group were dosed with E2-GS10-I1, formulated in PBS,at 10 and 100 mg/kg, ip and plasma samples were collected in citratephosphate dextrose solution at pre dosing, 0.5, 2, 4, 8, 12, 24, 48, 72,96, 144, and 168 hours post dosing. Plasma samples were assessed forpegylated E2-GS10-I1 Fn3-based binder levels using a quantitativeelectrochemiluminescence (ECL) assay developed to detect and quantitatethe pegylated E/I ¹⁰Fn3-based binder in plasma samples. In this assay, amouse monoclonal antibody with specificity toward the EGFR bindingregion was adsorbed to Meso Scale Discovery plates overnight at 4° C. toallow capture of the pegylated E/I ¹⁰Fn3-based binder in the plasmasamples. The plasma samples were added to the plates and incubated at22° C. for 1 h. The captured pegylated E/I ¹⁰Fn3-based binder wasdetected by a rabbit polyclonal antibody specific to the scaffold regionof the E/I ¹⁰Fn3-based binder, mixed with a goat anti-rabbit antibodylinked with a SULFO-TAG. Following a wash to remove unbound SULFO-TAGreagent, a read buffer was added and ECL detection was used. The levelof pegylated E2-GS10-I1 in plasma samples was calculated based oncomparison to a 4-parameter fit of a standard curve of the pegylatedE2-GS10-I1 Fn3-based binder.

Mice administered 10 or 100 mg/kg interperitoneally (ip) of pegylatedE2-GS10-I1 resulted in peak levels of approximately 200 and 1700 μg/mL,respectively, indicating dose-proportional pharmacokinetics (FIG. 30).Pharmacokinetic parameters for FIG. 30 were calculated in a similarfashion to those described in the paragraph below (note that “T ½” isinterchangeable with “HL_lambda_z” and AUC is interchangeable with“AUCINF_obs”. The half-life of pegylated E2-GS10-I1 in mice was15.75±1.52 h (FIG. 30). Based on these pharmacokinetic parameters,administration of 100 mg/kg three times weekly (TIW) in human tumorxenograft studies was able to maintain drug levels 10- to 100-foldhigher than the in vitro IC50 value.

Additional pharmacokinetic experiments were conducted on severalpegylated E/I ¹⁰Fn3-based binders, where mice were administered 10 or100 mg/kg interperitoneally (ip) and, for the pegylated I1-GS10-E5, 10or 64 mg/kg sub-cutaneously (sc), plasma was collected and analyzed asdescribed above to measure the levels of pegylated E/I ¹⁰Fn3-basedbinders. The pharmacokinetic parameters of these various E/I ¹⁰Fn3-basedbinders were obtained by non-compartmental analysis of plasma (serum)concentration vs. time data. WinNonlin software (version 5.1, PharsightCorp. Mountain View Calif.) was used to calculate the terminal half-life(HL_lambda_z), maximum observed concentration (Cmax), the area under thecurve from time zero extrapolated to infinity (AUCINF_obs), clearance(CL_F_obs), volume of distribution based on the terminal phase(Vz_F_obs) and the mean residence time extrapolated to infinity(MRTINF_obs). Results showed that the half life for the pegylated E/I¹⁰Fn3-based binders were between 12.1-20.9 hours, as shown in FIG. 44and FIG. 31.

Example 31 Pharmacodynamics

Samples were taken from the H292 and the DiFi xenograft models describedin Table 21 at the end of the study and processed as outlined underMeasurement of pharmacodynamic endpoints in tumors in Example 12 foranalysis of total levels of EGFR and IGFR protein and phosphorylatedEGFR and IGFR. Target effects of I1-GS10-E5-pegylated and panitumumabwere evaluated by immunoblotting as described in Example 11. In FIG.33A, levels of total EGFR, pEGFR and total IGFR were lower inI1-GS10-E5-pegylated treated tumors than in untreated tumors at the endof the DiFi xenograft model. In FIG. 33B, levels of pEGFR were lower intumors treated with panitumumab and I1-GS10-E5-pegylated. Levels oftotal EGFR were lower only in I1-GS10-E5-pegylated treated tumors butnot in panitumumab treated tumors. Levels of total IGFR were lower inboth I1-GS10-E5-pegylated treated tumors and in one panitumumab treatedtumor but not the other. The amount of pIGFR in these models was too lowto detect differences following treatment. Immunoblots were probed withGAPDH to demonstrate equal loading of protein.

Example 32 EGFR ¹⁰Fn3-Based Binders Optimization and Consensus SequenceAnalysis

The ¹⁰Fn3-based binder 679F09 (as described in PCT WO 2009/102421) (FIG.34) was identified as a binder to EGFR ectodomain-Fc fusion protein (R&DSystems). Binding activity was selected using a bead coated with EGFR-Fcand ¹⁰Fn3-based binders coupled to their nucleic acid coding sequence(see e.g., Xu et al., Directed Evolution of High-Affinity AntibodyMimics Using mRNA Display, Chem. Biol. 9: 933-942 (2002)). More potentvariants of the parental EGFR binder 679F09 having alterations to theamino acid sequences in the BC, DE and FG loops were also identified.

Sequence Analysis I: All ¹⁰Fn3-Based Binders Selected for High-AffinityBinding to EGFR

In order to reveal sequence patterns that defined strong affinity forEGFR, all unique EGFR binding sequences (1044) were analyzed usingseveral methods. First, the sequences were analyzed by the frequency ofamino acids at each position in the loops (FIGS. 35-38). Only uniquesequences for each loop were analyzed.

From the above sequence analysis, the following broad sequence motif wasdefined:

Sequence Motif #1

-   -   (a) BC loop: “YQ” in positions 7-8 (i.e., corresponding to        positions 29 and 30 of SEQ ID NO: 1)    -   (b) DE loop: aliphatic residue (“V/I/L/M/A”) in position 3        (i.e., corresponding to position 54 of SEQ ID NO: 1)    -   (c) FG loop: “D/N” in position 1 (i.e., corresponding to        position 77 of SEQ ID NO: 1)

All 1044 sequences analyzed, except one, follow the FG loop sequencepattern (c). Of all unique sequences analyzed, 90% follow pattern (a)for the BC loop, and 95% follow pattern (b) for the DE loop. Allsequences analyzed, except four, follow at least two of the threepatterns above. In addition, the 15-amino acid FG loop length is anoteworthy sequence feature.

In addition to the broad Sequence Motif #1 defined above, the data inFIGS. 35-38 were used to define a second sequence motif based on thedominant residues at each position. Residues were included in this motifif the sum of the top 3 most frequent amino acids had a greater than 50%frequency.

Sequence Motif #2

-   -   (a) BC loop: XXXXXXYQ (same as Motif #1), wherein X is any amino        acid    -   (b) DE loop: (G/Y/H)(D/M/G)(V/L/I)X, wherein X is any amino acid    -   (c) FG loop, 10 amino acid length:        (D/N)(Y/M)(Y/A/M)(Y/H/F)(K/Q/V)(E/P/R)(Y/T/K)X(E/Y/Q)(Y/G/H),        wherein X is any amino acid    -   (d) FG loop, 15 amino acid length:        D(Y/F/W)(Y/F/K)(N/D/P)(P/H/L)(A/T/V)(T/D/S)(H/Y/G)(E/P/V)(Y/H)(T/K/I)(Y/F)(H/N/Q)(T/Q/E)(T/S/I)

The analysis methods used to define Sequence Motifs #1 and #2 evaluateeach residue position within a loop separately. To reveal any sequencemotifs spanning multiple residues within a loop, the ¹⁰Fn3-based binderswere subjected to further analysis. In this analysis, the loop sequenceswere aligned using ClustalW (Thompson J D et al. CLUSTAL W: improvingthe sensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weight matrixchoice. Nucleic Acids Research 22: 4673-4680, 1994). From thisalignment, families of sequences were grouped using manual inspection.For the BC and DE loops, sequence patterns similar to Sequence Motifs #1and #2 were observed. However, additional sequence motifs could bedefined for the 10 and 15 amino acid long FG loops.

Sequence Motif #3

-   -   (a) FG loop, 10 amino acid length        -   (1) DY(A/Y)GKPYXEY (SEQ ID NO: 473), wherein X is any amino            acid        -   (2) DY(A/Y)Y(K/R/Q/T)PYXEY (SEQ ID NO: 474), wherein X is            any amino acid        -   (3) (D/N)Y(A/Y)(Y/F)(K/R/Q/T)EYXE(Y/H) (SEQ ID NO: 475),            wherein X is any amino acid        -   (4) DYY(H/Y)X(R/K)X(E/T)YX (SEQ ID NO: 476), wherein X is            any amino acid        -   (5) DYY(H/Y)(K/H/Q)(R/K)T(E/T)Y(G/P) (SEQ ID NO: 477)        -   (6) (D/N)MMHV(E/D)YXEY (SEQ ID NO: 478), wherein X is any            amino acid        -   (7) DYMHXXYXEY (SEQ ID NO: 479) (like FG loop of 679F09),            wherein X is any amino acid        -   (8) D(M/Y)YHX(K/R)X(V/I/L/M)YG (SEQ ID NO: 480), wherein X            is any amino acid    -   (b) FG loop, 15 amino acid length        -   (1) D(Y/F)(Y/F)NPXTHEYXYXXX (SEQ ID NO: 481), wherein X is            any amino acid        -   (2) D(Y/F)(Y/F)D(P/L)X(T/S)HXYXYXXX (SEQ ID NO: 482),            wherein X is any amino acid        -   (3) D(Y/F)(K/R)PHXDGPH(T/I)YXE(S/Y) (SEQ ID NO: 483),            wherein X is any amino acid

Sequence Analysis II: ¹⁰Fn3-Based Binders Showing More Potent Inhibitionof EGFR Phosphorylation

Another overall sequence analysis was performed on the subset of¹⁰Fn3-based binders that showed the most potent activity in a cell-basedassay (as opposed to Sequence Analysis I, which was performed on allbinders selected for high-affinity binding to EGFR through Profusion).Because many of the binders were only run through single-pointcell-based assays, binders that showed greater than 75% inhibition ofEGFR phosphorylation at a fixed concentration of 100 nM were included inthis analysis. The percent inhibition at a given concentration isrelated to the IC50 by: % inhibition=100×concentration/(concentration+IC50).

Normally, an IC50 is calculated by fitting the data for % inhibition atvarious concentrations. However, given that only a single data point isavailable for each binder, it is inappropriate to use this single datapoint to calculate an IC50. Therefore, the percent inhibition of EGFRsignaling at a single concentration point was used as an approximationof the potency of the binder. Although a binder may show 75% inhibitionat a concentration of 100 nM, increasing the concentration will allowthe clone to show 100% inhibition at a higher concentration. The %inhibition is inversely related to the IC50; i.e., the higher the %inhibition, the lower the IC50 and the more potent the binder. If abinder showed 75% inhibition at a concentration of 100 nM, we consideredthis to be a “potent” binder for the purposes of Sequence Analysis II.However, the binders which showed less than 75% inhibition at 100 nMconcentration for the most part still bind to EGFR and still have aneffect on EGFR signaling. For instance, the anti-EGFR monoclonalantibody Nimotuzumab (Friedlander E et al. ErbB-directed immunotherapy:antibodies in current practice and promising new agents. Immunol Lett116: 126-140, 2008) is currently under development as a therapeutic, butit shows <5% inhibition at a 100 nM concentration in the EGFRphosphorylation assay (data not shown). The sequences of all “potent”binders assayed and their % inhibition of EGFR phosphorylation at 100 nMconcentration is shown in FIG. 45.

The total number of unique ¹⁰Fn3-based binders that showed >75%inhibition at 100 nM concentration was 111. As before, the sequencesfirst were analyzed by the frequency of amino acids at each position inthe loops (FIGS. 39-42). Since these binders are a subset of all thebinders selected for high affinity binding to EGFR during Profusion,they also follow Sequence Motif #1 (see above). All “potent” sequencesanalyzed follow the FG loop sequence pattern (“D/N” in position 1). Ofall unique “potent” sequences analyzed, 93% follow the pattern for theBC loop (“YQ” in positions 7-8), and 98% follow the pattern for the DEloop (aliphatic residue (“V/I/L/M/A”) in position 3). All “potent”sequences analyzed follow at least two of the three patterns of SequenceMotif #1.

Of note, the 15-amino acid FG loop length also appears to be highlyrepresented in the most “potent” binders. While 15-amino acid long FGloops represent only 55% of all binders selected for high affinitybinding to EGFR (Sequence Analysis I), 15-amino acid FG loops represent86% of the binders with >50% inhibition of EGFR phosphorylation at 100nM concentration, and 91% of the binders with >75% inhibition (“potent”binders in Sequence Analysis II). Therefore, the longer 15-amino acid FGloop appears to be a sequence pattern associated with greater potency.

Of the 111 “potent” sequences analyzed, only 10 contain 10-amino acidlong FG loops, and 6 of those are unique. Therefore, a single sequencemotif can encompass every “potent” 10-amino acid FG loop sequence.Sequence Motif #4 was defined based on these 6 sequences.

Sequence Motif #4

-   -   FG loop, 10-amino acid length, “potent” binders    -   (D/N)(M/Y)(M/A/W)(H/F/Y)(V/K)EY(A/Q/R/S/T)E(Y/H/D)

The sequence analysis of the “potent” binders with 15-amino acid FGloops also further illuminated which residue positions were mostconserved, allowing Sequence Motif #5 to be defined. An “X” in thissequence motif denotes positions where there are no three dominant aminoacids.

Sequence Motif #5

-   -   FG loop, 15-amino acid length, “potent” binders    -   D(Y/F/W)(Y/F/K)(N/P/D)(P/H/L)X(T/D/S)(H/G/Y)(E/P/Y)(Y/H)XYXXX,        wherein X is any amino acid

All of the EGFR binders that were analyzed are progeny of the parent679F09 and constitute a sequence “family,” i.e. they are all related insequence according to the aforementioned sequence motifs. Variousmembers of the 679F09 family of binders can tolerate a T51I scaffoldmutation and retain binding activity. Therefore, a T511 scaffoldmutation could be combined with any of the aforementioned sequencemotifs to also yield a binder with high affinity binding to EGFR.

Finally, it should be noted that amino acids with similar properties canoften be substituted into protein sequences with little or no effect onstructure or function. This indeed is the case for ¹⁰Fn3-based bindersas well, where conservative amino acid substitutions in either the loopor scaffold regions can still lead to binders which bind to EGFR. Forinstance, substituting “Y” for “H” in the second position of the FG loopof binder E98 yields binder E99, and both binders show similar potencyin inhibiting EGFR phosphorylation (FIG. 45).

We claim:
 1. An antibody-like protein comprising a tenth fibronectintype III domain (¹⁰Fn3) that binds epidermal growth factor receptor(EGFR) with a K_(D) of less than 500 nM, wherein the EGFR binding ¹⁰Fn3comprises a BC loop having the amino acid sequence set forth in aminoacids 21-30 of SEQ ID NO: 112, a DE loop having the amino acid sequenceset forth in amino acids 51-56 of SEQ ID NO: 112, and an FG loop havingthe amino acid sequence set forth in amino acids 76-92 of SEQ ID NO:112.
 2. The antibody-like protein of claim 1, further comprising asecond tenth fibronectin type III domain (10Fn3).
 3. The antibody-likeprotein of claim 2, wherein the second ¹⁰Fn3 is covalently linked to theEGFR binding ¹⁰Fn3 via a polypeptide linker or a polyethylene glycolmoiety.
 4. A pharmaceutically acceptable composition comprising theantibody-like protein of claim 1, wherein the composition is essentiallypyrogen free.
 5. The antibody-like protein of claim 1, comprising anamino acid sequence at least 90% identical to SEQ ID NO:
 112. 6. Theantibody-like protein of claim 5, comprising an amino acid sequence atleast 95% identical to SEQ ID NO:
 112. 7. The antibody-like protein ofclaim 6, comprising an amino acid sequence at least 98% identical to SEQID NO:
 112. 8. An antibody-like protein comprising the amino acidsequence of SEQ ID NO:
 112. 9. An antibody-like protein comprising atenth fibronectin type III domain (¹⁰Fn3) that binds epidermal growthfactor receptor (EGFR) with a K_(D) of less than 500 nM, wherein theEGFR binding ¹⁰Fn3 comprises a BC loop having the amino acid sequenceX_(g)WAPVDRYQX_(h) (SEQ ID NO: 137), a DE loop having the amino acidsequence X_(i)RDVYX_(J) (SEQ ID NO: 138), and an FG loop having theamino acid sequence X_(k)DYKPHADGPHTYHESX_(l)(SEQ ID NO: 139); andwherein X is any amino acid and g, h, i, j, k, and l are integersindependently selected from 0 to
 5. 10. The antibody-like protein ofclaim 9, wherein the EGFR binding ¹⁰Fn3 comprises a BC loop having theamino acid sequence SWWAPVDRYQ (SEQ ID NO: 115), a DE loop having theamino acid sequence PRDVYT (SEQ ID NO: 116), and an FG loop having theamino acid sequence TDYKPHADGPHTYHESP (SEQ ID NO: 117).